Polypeptides and nucleic acids encoding same

ABSTRACT

DNAs are provided, whose genes are induced at least by Wnt-1. Also provided are nucleic acid molecules encoding those polypeptides, as well as vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides, and methods for producing the polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.10/628,770, filed Jul. 28, 2003, which application is a continuation ofU.S. Ser. No. 09/182,562, filed Oct. 29, 1998, now abandoned, claimingpriority to provisional application Ser. No. 60/063,704, filed Oct. 29,1997, and to provisional application Ser. No. 60/073,612, filed Feb. 4,1998, the entire disclosures of which applications are herebyincorporated by reference.

GOVERNMENT INTERESTS

This invention was made with government support under grant no. 5PO1CA41086, awarded by the National Institutes of Health, National CancerInstitute. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides useful in the management of malignancies.

BACKGROUND OF THE INVENTION

Wnts are encoded by a large gene family whose members have been found inround worms, insects, cartilaginous fish, and vertebrates. Holland etal., Dev. Suppl., 125-133 (1994). Wnts are thought to function in avariety of developmental and physiological processes since many diversespecies have multiple conserved Wnt genes. McMahon, Trends Genet., 8:236-242 (1992); Nusse and Varmus, Cell, 69: 1073-1087 (1992). Wnt genesencode secreted glycoproteins that are thought to function as paracrineor autocrine signals active in several primitive cell types. McMahon,supra; Nusse and Varmus, supra. The Wnt growth factor family includesmore than ten genes identified in the mouse (Wnt-1, -2, -3A, -3B, -4,-5A, -5B, -6, -7A, -7B, -8A, -8B, -10B, -11, -12, and -13) (see, e.g.,Gavin et al., Genes Dev., 4: 2319-2332 (1990); Lee et al., Proc. Natl.Acad. Sci. USA, 92: 2268-2272 (1995); Christiansen et al., Mech. Dev.,51: 341-350 (1995)) and at least nine genes identified in the human(Wnt-1, -2, -3, -5A, -7A, -7B, -8B, -10B, and -11) by cDNA cloning. See,e.g., Vant Veer et al., Mol. Cell. Biol., 4: 2532-2534 (1984).

The Wnt-1 proto-oncogene (int-1) was originally identified from mammarytumors induced by mouse mammary tumor virus (MMTV) due to an insertionof viral DNA sequence. Nusse and Varmus, Cell, 31: 99-109 (1982). Inadult mice, the expression level of Wnt-1 mRNA is detected only in thetestis during later stages of sperm development. Wnt-1 protein is about42 KDa and contains an amino-terminal hydrophobic region, which mayfunction as a signal sequence for secretion. Nusse and Varmus, supra,1992. The expression of Wnt-2/irp is detected in mouse fetal and adulttissues and its distribution does not overlap with the expressionpattern for Wnt-1. Wnt-3 is associated with mouse mammary tumorigenesis.The expression of Wnt-3 in mouse embryos is detected in the neural tubesand in the limb buds. Wnt-5a transcripts are detected in the developingfore- and hind limbs at 9.5 through 14.5 days and highest levels areconcentrated in apical ectoderm at the distal tip of limbs. Nusse andVarmus, supra (1992). Recently, a Wnt growth factor, termed Wnt-x, wasdescribed (WO95/17416) along with the detection of Wnt-x expression inbone tissues and in bone-derived cells. Also described was the role ofWnt-x in the maintenance of mature osteoblasts and the use of the Wnt-xgrowth factor as a therapeutic agent or in the development of othertherapeutic agents to treat bone-related diseases.

Wnts may play a role in local cell signaling. Biochemical studies haveshown that much of the secreted Wnt protein can be found associated withthe cell surface or extracellular matrix rather than freely diffusiblein the medium. Papkoff and Schryver, Mol. Cell. Biol., 10: 2723-2730(1990); Bradley and Brown, EMBO J., 9: 1569-1575 (1990).

Studies of mutations in Wnt genes have indicated a role for Wnts ingrowth control and tissue patterning. In Drosophila, wingless (wg)encodes a Wnt-related gene (Rijsewik et al., Cell, 50: 649-657 (1987))and wg mutations alter the pattern of embryonic ectoderm, neurogenesis,and imaginal disc outgrowth. Morata and Lawerence, Dev. Biol., 56:227-240 (1977); Baker, Dev. Biol., 125: 96-108 (1988); Klingensmith andNusse, Dev. Biol., 166: 396-414 (1994). In Caenorhabditis elegans,lin-44 encodes a Wnt homolog which is required for asymmetric celldivisions. Herman and Horvitz, Development, 120: 1035-1047 (1994).Knock-out mutations in mice have shown Wnts to be essential for braindevelopment (McMahon and Bradley, Cell, 62: 1073-1085 (1990); Thomas andCappechi, Nature, 346: 847-850 (1990)), and the outgrowth of embryonicprimordia for kidney (Stark et al., Nature, 372: 679-683 (1994)), tailbud (Takada et al., Genes Dev., 8: 174-189 (1994)), and limb bud. Parrand McMahon, Nature, 374: 350-353 (1995). Overexpression of Wnts in themammary gland can result in mammary hyperplasia (McMahon, supra (1992);Nusse and Varmus, supra (1992)), and precocious alveolar development.Bradbury et al., Dev. Biol., 170: 553-563 (1995).

Wnt-5a and Wnt-5b are expressed in the posterior and lateral mesodermand the extraembryonic mesoderm of the day 7-8 murine embryo. Gavin etal., supra. These embryonic domains contribute to the AGM region andyolk sac tissues from which multipotent hematopoietic precursors andHSCs are derived. Dzierzak and Medvinsky, Trends Genet., 11: 359-366(1995); Zon et al., in Gluckman and Coulombel, ed., Colloque, INSERM,235: 17-22 (1995), presented at the Joint International Workshop onFoetal and Neonatal Hematopoiesis and Mechanism of Bone Marrow Failure,Paris France, Apr. 3-6, 1995; Kanatsu and Nishikawa, Development, 122:823-830 (1996). Wnt-5a, Wnt-10b, and other Wnts have been detected inlimb buds, indicating possible roles in the development and patterningof the early bone microenvironment as shown for Wnt-7b. Gavin et al.,supra; Christiansen et al., Mech. Devel., 51: 341-350 (1995); Parr andMcMahon, supra.

The Wnt/Wg signal transduction pathway plays an important role in thebiological development of the organism and has been implicated inseveral human cancers. This pathway also includes the tumor suppressorgene, APC. Mutations in the APC gene are associated with the developmentof sporadic and inherited forms of human colorectal cancer. The Wnt/Wgsignal leads to the accumulation of beta-catenin/Armadillo in the cell,resulting in the formation of a bipartite transcription complexconsisting of beta-catenin and a member of the lymphoid enhancer bindingfactor/T cell factor (LEF/TCF)HMG box transcription factor family. Thiscomplex translocates to the nucleus where it can activate expression ofgenes downstream of the Wnt/Wg signal, such as the engrailed andUltrabithorax genes in Drosophila. The downstream target genes of Wnt-1signaling in vertebrates that presumably function in tumorigenesis,however, are currently unknown.

For a most recent review on Wnt, see Cadigan and Nusse, Genes & Dev.,11: 3286-3305 (1997).

Another family of proteins, the Rho and Rae subfamilies of Ras proteins,have been implicated in transformation by oncogenic ras. Thus far,activation of the pathways governed by three members of the Rho familyof GTP-binding proteins, CDC42, Rac, and Rho, has been found to benecessary for Ras transformation. Activating Ras mutations occur inabout 30% of all human tumors, indicating that elements of the CDC42,Rac, and Rho signaling pathways are drug targets for cancer therapy.These three members play a central role in the organization of the actincytoskeleton and regulate transcription. Like Ras, the Rho proteinsinteract directly with protein kinases, which are likely to serve asdownstream effector targets of the activated GTPase. The roles of thedifferent Rho proteins in Ras transformation appear to be distinct:CDC42 specifically controls anchorage-independent growth, whereas Raecontrols Rac-induced mitogenicity. The small G proteins Rac1, Rac2, andRac3 are highly related GTPases. Didsbury et al., J. Biol. Chem., 264:16378-16382 (1989); Moll et al., Oncogene, 6: 863-866 (1991); Shirsat etal., Oncogene, 5: 769-772 (1990); Haataja et al., J. Biol. Chem., 272:20384-20388 (1997). RAC3 is located at chromosome 17q23-25, a regionfrequently deleted in breast cancer. Cropp et al., Proc. Natl. Acad.Sci. USA 87: 7737-7741 (1990); Cornelis et al., Oncogene, 8: 781-785(1993). Recent data have provided evidence that constitutive activity ofthe Rho-family GTPases is associated with cytoskeletal rearrangement anddisorganized growth, motility, and invasiveness of cells, all hallmarksof neoplasia.

There is a need to elucidate the further members of the above families,including cell-surface molecules that may be tumor-specific antigens orproteins that serve a regulatory function in initiating the Wnt pathwayof tumorigenesis. These would also include downstream components of theWnt signaling pathway that are important to the transformed phenotypeand the development of cancer. There is also a need to identify otherproteins that, perhaps in conjunction with beta-catenin, regulate Wnt-1downstream genes, as well as GTPases.

SUMMARY OF THE INVENTION

Several putative Wnt-1-induced genes have been identified at the mRNAlevel in a high-throughput cDNA substraction experiment. Thus,applicants have identified novel cDNA clones (clone 65 and clone 320)that encode novel polypeptides that are Wnt induced, designated as clone65 and clone 320, respectively. The clone 65 molecules have homology tothe Rae and Rho subfamily noted above.

In one embodiment, this invention provides isolated nucleic acidcomprising DNA having at least about 800 nucleotides and at least abouta 70% sequence identity to (a) a DNA molecule encoding a human clone 65polypeptide comprising the sequence of amino acids 1 to 258 of FIGS. 5Aand 5B (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a).Preferably, this nucleic acid has at least one clone 65 or 320biological activity.

In another aspect, the invention provides isolated nucleic acidcomprising DNA having at least about 700 nucleotides and at least abouta 95% sequence identity to (a) a DNA molecule encoding a human clone 65polypeptide comprising the sequence of amino acids 1 to 258 of FIGS. 5Aand 5B (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a).Preferably, this nucleic acid comprises DNA encoding a human clone 65polypeptide having amino acid residues 1 to 258 of FIGS. 5A and 5B (SEQID NO:3), or the complement thereof. In a still further aspect, theinvention provides isolated nucleic acid comprising DNA having at leastabout 800 nucleotides and at least about a 70% sequence identity to (a)a DNA molecule encoding a mouse clone 65 polypeptide comprising thesequence of amino acids 1 to 261 of FIGS. 1A and 1B (SFQ ID NO:6), or(b) the complement of the DNA molecule of (a). Preferably, this nucleicacid comprises DNA having at least about a 85% sequence identity to (a)a DNA molecule encoding a mouse clone 65 polypeptide comprising thesequence of amino acids 1 to 261 of FIGS. 1A and 1B (SEQ ID NO:6), or(b) the complement of the DNA molecule of (a). Preferably, this nucleicacid comprises DNA encoding a mouse clone 65 polypeptide having aminoacid residues 1 to 261 of FIGS. 1A and 1B (SEQ ID NO:6), or thecomplement thereof.

In a still further embodiment, the invention provides an isolatednucleic acid comprising DNA having at least about 800 nucleotides and atleast about a 70% sequence identity to (a) a DNA molecule encoding thesame full-length polypeptide encoded by the human clone 65 polypeptidecDNA in ATCC Deposit No. 209536 (pRK5E.h.WIG-3.65.4A), or (b) thecomplement of the DNA molecule of (a). Preferably, this nucleic acidcomprises DNA having at least about a 95% sequence identity to (a) a DNAmolecule encoding the same full-length polypeptide encoded by the humanclone 65 polypeptide cDNA in ATCC Deposit No. 209536(pRK5E.h.WIG-3.65.4A), or (b) the complement of the DNA molecule of (a).

In another embodiment, the invention provides isolated nucleic acidcomprising SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, or 19, and anisolated clone 65 polypeptide encoded by such a nucleic acid.

A still further aspect of the invention involves a process for producinga clone 65 polypeptide comprising culturing a host cell comprising clone65-encoding nucleic acid under conditions suitable for expression of theclone 65 polypeptide and recovering the clone 65 polypeptide from thecell culture.

Further provided is an isolated clone 65 polypeptide encoded by theclone 65-encoding nucleic acid. Preferably, this polypeptide is humanclone 65 or mouse clone 65.

In another embodiment, the invention provides an isolated nucleic acidhaving at least about 800 nucleotides and produced by hybridizing a testDNA molecule under stringent conditions with (a) a DNA molecule encodinga human clone 65 polypeptide comprising the sequence of amino acids 1 to258 of FIGS. 5A and 5B (SEQ ID NO:3), or (b) the complement of the DNAmolecule of (a), and, if the test DNA molecule has at least about a 70%sequence identity to (a) or (b), isolating the test DNA molecule.

Also provided is a polypeptide produced by (i) hybridizing a test DNAmolecule under stringent conditions with (a) a DNA molecule encoding ahuman clone 65 polypeptide comprising the sequence of amino acids 1 to258 of FIGS. 5A and 5B (SEQ ID NO:3), or (b) the complement of the DNAmolecule of (a), and if the test DNA molecule has at least about a 70%sequence identity to (a) or (b), (ii) culturing a host cell comprisingthe test DNA molecule under conditions suitable for expression of thepolypeptide, and (iii) recovering the polypeptide from the cell culture.

Also provided by the invention is isolated nucleic acid encoding mouseclone 320 comprising DNA having at least about 500 nucleotides andhaving at least about a 97% sequence identity to (a) a DNA moleculecomprising the sequence of nucleotides 1 to 2822 of FIG. 2 (SEQ IDNO:7), or (b) the complement of the DNA molecule of (a). Furtherprovided is isolated nucleic acid encoding mouse clone 320 comprisingDNA having at least about 700 nucleotides and having at least about a70% sequence identity, more preferably at least about an 80% sequenceidentity, more preferably still at least about a 90% sequence identity,and yet more preferably at least about a 95% sequence identity, and morepreferably at least about a 100% sequence identity, to (a) a DNAmolecule comprising the sequence of nucleotides 1 to 727 of FIG. 3 (SEQID NO:8), or (b) the complement of the DNA molecule of (a). Alsoprovided is isolated nucleic acid encoding mouse clone 320 comprisingDNA having at least about 700 nucleotides and having at least 75%sequence identity to (a) a DNA molecule comprising the sequence ofnucleotides 1-2526 of FIGS. 4A and 4B (SEQ ID NO:9), or (b) thecomplement of the DNA molecule of (a). More preferably, this nucleic hasat least about 90% sequence identity to (a) or (b).

Also provided by the invention is isolated nucleic acid encoding mouseclone 320 comprising DNA having at least about 500 nucleotides andhaving at least about a 97% sequence identity to (a) a DNA moleculecomprising the sequence of nucleotides 1 to 2822 of FIG. 2 (SEQ IDNO:7), or (b) the complement of the DNA molecule of (a) and comprisingDNA having at least about 700 nucleotides and having at least about a70% sequence identity, more preferably at least about an 80% sequenceidentity, more preferably still at least about a 90% sequence identity,and yet more preferably at least about a 95% sequence identity, and morepreferably at least about a 100% sequence identity, to (c) a DNAmolecule comprising the sequence of nucleotides 1 to 727 of FIG. 3 (SEQID NO:8), or (d) the complement of the DNA molecule of (c).

Further provided is isolated nucleic acid encoding mouse clone 320comprising DNA having at least about 500 nucleotides and having at leastabout a 97% sequence identity to (a) a DNA molecule comprising thesequence of nucleotides 1 to 2822 of FIG. 2 (SEQ ID NO:7), or (b) thecomplement of the DNA molecule of (a) and comprising DNA having at leastabout 700 nucleotides and having at least a 75% sequence identity to (c)a DNA molecule comprising the sequence of nucleotides 1 to 2526 of FIGS.4A and 4B (SEQ ID NO: 9), or (d) the complement of the DNA molecule of(c).

Additionally provided is isolated nucleic acid encoding mouse clone 320comprising DNA having at least about 700 nucleotides and having at leastabout a 70% sequence identity to (a) a DNA molecule comprising thesequence of nucleotides 1 to 727 of FIG. 3 (SEQ ID NO:8), or (b) thecomplement of the DNA molecule of (a) and comprising DNA having at leastabout 700 nucleotides and having at least a 75% sequence identity to (c)a DNA molecule comprising the sequence of nucleotides 1 to 2526 of FIGS.4A and 4B (SEQ ID NO: 9), or (d) the complement of the DNA molecule of(c).

Still further provided is isolated nucleic acid encoding mouse clone 320comprising DNA having at least about 500 nucleotides and having at leastabout a 97% sequence identity to (a) a DNA molecule comprising thesequence of nucleotides 1 to 2822 of FIG. 2 (SEQ ID NO:7), or (b) thecomplement of the DNA molecule of (a) and comprising DNA having at leastabout 700 nucleotides and having at least a 75% sequence identity to (c)a DNA molecule comprising the sequence of nucleotides 1 to 2526 of FIGS.4A and 4B (SEQ ID NO: 9), or (d) the complement of the DNA molecule of(c) and comprising DNA having at least about 700 nucleotides and havingat least about a 70% sequence identity to (e) a DNA molecule comprisingthe sequence of nucleotides 1-727 of FIG. 3 (SEQ ID NO:8), or (f) thecomplement of the DNA molecule of (e).

Also provided by the invention is isolated nucleic acid encoding mouseclone 320 comprising DNA having at least about 500 nucleotides andhaving at least about a 95% sequence identity to (a) a DNA moleculeencoding the same polypeptide encoded by the mouse clone 320 polypeptidecDNA in ATCC Deposit No. 209534_ (pRK5E.m.WIG-4.320.9), or (b) thecomplement of the DNA molecule of (a). Preferably, the nucleic acid isabout 3 kilobases in length.

More preferably, the nucleic acid comprises DNA that encodes the samepolypeptide encoded by the mouse clone 320 polypeptide cDNA in ATCCDeposit No. 209534 (pRK5E.m.WIG-4.320.9).

In another aspect, the invention provides a polypeptide encoded by theclone 320 nucleic acid above.

Further provided is an isolated nucleic acid having at least about 500nucleotides and produced by hybridizing a test DNA molecule understringent conditions with (a) a DNA molecule encoding the samepolypeptide encoded by the mouse clone 320 polypeptide cDNA in ATCCDeposit No. 209534 (pRK5E.m.WIG-4.320.9), or (b) the complement of theDNA molecule of (a), and, if the test DNA molecule has at least about a95% sequence identity to (a) or (b), isolating the test DNA molecule. Ina still further embodiment, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding the same polypeptide encodedby the mouse clone 320 polypeptide cDNA in ATCC Deposit No. 209534(pRK5E.m.WIG-4.320.9), or (b) the complement of the DNA molecule of (a),and if the test DNA molecule has at least about a 95% sequence identityto (a) or (b), (ii) culturing a host cell comprising the test DNAmolecule under conditions suitable for expression of the polypeptide,and (iii) recovering the polypeptide from the cell culture. Preferablythe complements of the DNA molecules herein remain stably bound to theprimary sequence under at least moderate, and optionally, under highstringency conditions.

Also provided are vectors comprising the above nucleic acids, host cellscomprising the vector, preferably wherein the cell is a Chinese hamsterovary (CHO) cell, an E. coli cell, a baculovirus-infected cell, or ayeast cell.

Additionally provided are a chimeric molecule comprising one of theabove polypeptides or an inactivated variant thereof, fused to aheterologous amino acid sequence, wherein the heterologous amino acidsequence may be, for example, an epitope tag sequence or an Fc region ofan immunoglobulin. Also provided is an antibody which specifically bindsto one of the above polypeptides, wherein the antibody can be amonoclonal antibody. Further provided are a composition comprising oneof the above polypeptides and a carrier therefor, and a compositioncomprising an antagonist to one of the polypeptides and a carriertherefor. Preferably, these compositions may also comprise achemotherapeutic agent or growth-inhibitory agent.

In another embodiment, the invention provides a method for treating aclone 65- or 320-related disorder in a mammal comprising administeringto the mammal an effective amount of any of the above compositions.Preferably, the disorder is a malignant disorder or arteriosclerosis.More preferably, the malignant disorder is breast cancer, ovariancancer, colon cancer, or melanoma.

Also provided herein is a kit comprising one of the above clone 65 or320 polypeptides or clone 65 or 320 antagonists, such as anti-clone 65antibodies or anti-clone 320 antibodies, and instructions for using theantibody to detect a cancer induced by Wnt. Also provided is a methodfor inducing cell death comprising exposing a cell which is induced byWnt to an effective amount of one of the above clone 65 or 320polypeptides or clone 65 or 320 antagonists, such as anti-clone 65 oranti-clone 320 antibodies. Preferably, such cell is a cancer cell. Morepreferably, the cell is in a mammal, more preferably a human.Optionally, an effective amount of another chemotherapeutic antibody isalso exposed to the cell, such as an anti-ErbB2 antibody. Further,optionally the method comprises exposing the cell to a chemotherapeuticagent, a growth-inhibitory agent, or radiation. Optionally, the cell isexposed to the growth-inhibitory agent prior to exposure to theanti-clone 65 or anti-clone 320 antibody.

In a further aspect, the invention provides an article of manufacture,comprising:

a container;

a label on the container; and

a composition comprising an active agent contained within the container;wherein the composition is effective for inducing cell death, the labelon the container indicates that the composition can be used for treatingconditions characterized by overinduction of Wnt, and the active agentin the composition is one of the polypeptides noted above, or anantagonist to one of the polypeptides such as an antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the derived amino acid sequence of anative-sequence mouse clone 65 protein from amino acids 1 to 261 (SEQ IDNO:6) and the nucleotide sequence (and complementary sequence) encodingthe protein (SEQ ID NOS:4 and 5, respectively). This is from the mouseclone 65.11.3. A histidine sequence was fused to the heat-stableantigen. The heat-stable antigen sequence has been removed. There is 465bp of 3′ untranslated region and 86 bp of the coding region. The first139 bp of the sequence has 89% GC content. This is the added regioncompared to other family members. A potential glycosylation site is atamino acid 88 through 91. A potential protein kinase C phosphorylationsite is at amino acids 210 through 212. Potential casein kinase IIphosphorylation sites are at amino acids 84 through 87, 122 through 125,164 through 167, and 202 through 205. Potential N-myristoylation sitesare at amino acids 29 through 34, 37 through 42, 46 through 51, and 225through 240. A potential prenyl group binding site is at amino acids 258through 261. A potential ATP/GTP-binding site motif A (P-loop) is atamino acids 59 through 66.

FIG. 2 shows a nucleotide sequence (SEQ ID NO:7) contained within themouse clone 320 polypeptide cDNA in ATCC deposit no. 209534.

FIG. 3 shows another nucleotide sequence (SEQ ID NO:8) contained withinthe mouse clone 320 polypeptide cDNA in ATCC deposit no. 209534.

FIGS. 4A and 4B show yet another nucleotide sequence (and the complementthereof) (SEQ ID NOS:9 and 10, respectively) contained within the mouseclone

320 polypeptide cDNA in ATCC deposit no. 209534.

FIGS. 5A and 5B show the derived amino acid sequence of anative-sequence human clone 65 protein from amino acids 1 to 258 (SEQ IDNO:3) and the consensus nucleotide sequence (and complementary sequence)encoding the protein (SEQ ID NOS:1 and 2, respectively), which isderived from three human clones from a human fetal liver library. Thereare 2955 bp of 3′ untranslated region and 777 bp of coding region in thesequence. Potential N-glycosylation sites are from amino acids 85 though88, amino acids 138 through 141, and amino acids 245 through 248.Potential protein kinase C phosphorylation sites are at amino acids 140through 142 and 207 through 209. Potential casein kinase IIphosphorylation sites are at amino acids 81 through 84, 119 through 122,161 through 164, and 199 through 202. Potential N-myristoylation sitesare at amino acids 26 through 31, 43 through 48, and 222 through 227. Apotential ATP/GTP-binding site motif A (P-loop) is at amino acids 56through 63.

FIG. 6 shows an alignment of the full-length amino acid sequences of thehuman and mouse clone 65 (SEQ ID NOS:3 and 6, respectively).

FIG. 7 shows a map of the vector pBabe puro (5.1 kb) used to transformcells for purposes of differential expression. The vector includes bothunique restriction sites and multiple restriction sites. It is shownhere in modified form for Wnt-1 cloning wherein the HindIII site afterthe SV40 promoter in the original pBabe puro vector has been removed anda HindIII site added to the multiple cloning site of the original pBabepuro vector. Wnt-1 is cloned from EcoRI-HindIII in the multiple cloningsite. Constructs derived from this vector are selected in ampicillin(100 μg/ml) and the cells infected in culture are selected in 1.0-2.5μg/ml puromycin.

FIG. 8 shows the sequences of the PCR-Select® cDNA synthesis primer (SEQID NO:20), adaptors 1 and 2 (SEQ ID NOS:21 and 22, respectively) andcomplementary sequences for the adaptors (SEQ ID NOS:23 and 24,respectively), PCR primer 1 (SEQ ID NO:25), PCR primer 2 (SEQ ID NO:26),nested PCR primer 1 (SEQ ID NO:27), nested PCR primer 2 (SEQ ID NO:28),control primer G3PDH 5′ primer (SEQ ID NO:29), and control primer G3PDH3′ primer (SEQ ID NO:30) used for suppression subtractive hybridizationfor identifying clones 65 and 320. When the adaptors are ligated toRsaI-digested cDNA, the RsaI site is restored.

FIG. 9 shows the cloning site region of the plasmid pGEM-T used to cloneall of the clone 65 and 320 sequences herein (SEQ ID NOS:31 and 32 for5′ and 3′ sequences, respectively).

FIG. 10 shows the sequence (SEQ ID NO: 12) of a clone 65.11 obtained byscreening with a probe derived from clone 65, which is the initial cloneisolated in the process to obtain full-length mouse clone 65 DNA.

FIG. 11 shows the sequence (SEQ ID NO: 13) of a clone 65.9 obtained byscreening with a probe derived from clone 65.

FIG. 12 shows the sequence (SEQ ID NO: 14) of a clone 65.11.1 obtainedby screening with a probe derived from the CDC-42 homologous region ofclone 65.11.

FIGS. 13A and 13B show the sequence (SEQ ID NO:15) of a clone 65.11.3obtained by screening with a probe derived from the CDC-42 homologousregion of clone 65.11.

FIG. 14 shows the sequence (SEQ ID NO: 16) of a clone 65.11.6 obtainedby screening with a probe derived from the CDC-42 homologous region ofclone 65.11.

FIG. 15 shows the sequence (SEQ ID NO: 17) of clone 65.1 obtained byscreening with a probe derived from clone 65.

FIG. 16 shows the sequence (SEQ ID NO: 18) of clone 65.6 obtained byscreening with a probe derived from clone 65.

FIG. 17 shows the sequence (SEQ ID NO: 19) of clone 65.13 obtained byscreening with a probe derived from clone 65.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “clone 65 polypeptide”, “clone 65 homologue” and grammaticalvariants thereof, as used herein, encompass native-sequence proteinderived from clone 65 and variants thereof (which are further definedherein). The clone 65 polypeptide may be isolated from a variety ofsources, such as from human tissue types or from another source, orprepared by recombinant or synthetic methods, or by any combination ofthese and similar techniques.

The terms “clone 320 polypeptide”, “clone 320 homologue”, andgrammatical variants thereof, as used herein, encompass native-sequenceprotein derived from clone 320 and variants thereof (which are furtherdefined herein). The clone 320 polypeptide may be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant or synthetic methods, or by anycombination of these and similar techniques.

A “native-sequence clone 65 polypeptide” comprises a polypeptide havingthe same amino acid sequence as a clone 65 polypeptide derived fromnature. Such native-sequence clone 65 polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native-sequence clone 65 polypeptide” specifically encompassesnaturally-occurring truncated or other forms of a clone 65 polypeptidedisclosed herein, naturally-occurring variant forms (e.g.alternatively-spliced forms or splice variants), and naturally-occurringallelic variants of a clone 65 polypeptide. In one embodiment of theinvention, the native-sequence clone 65 polypeptide is a full-length,native-sequence human clone 65 polypeptide comprising amino acids 1 to258 of FIGS. 5A and 5B (SEQ ID NO:3), with or without the N-terminalmethionine. In another embodiment of the invention, the native-sequenceclone 65 polypeptide is a full-length native-sequence mouse clone 65polypeptide comprising amino acids 1 to 261 of FIGS. 1A and 1B (SEQ IDNO:6), with or without the N-terminal methionine.

In another embodiment of the invention, the native-sequence clone 65polypeptide is one which is encoded by a nucleotide sequence comprisingone of the mouse clone 65 splice or other native-sequence variants,including SEQ ID NOS:11, 12, 13, 14, 15, 16, 17, 18, or 19, with orwithout an N-terminal methionine.

A “native-sequence clone 320 polypeptide” comprises a polypeptide havingthe same amino acid sequence as a clone 320 polypeptide derived fromnature. Such native-sequence clone 320 polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native-sequence clone 320 polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of a clone 320polypeptide disclosed herein, naturally-occurring variant forms (e.g.,alternatively spliced forms or splice variants), and naturally-occurringallelic variants of a clone 320 polypeptide. In one embodiment of theinvention, the native-sequence clone 320 polypeptide is a mature orfull-length, native-sequence mouse clone 320 polypeptide comprising theinsert of about 3 kilobases from pRKSE.m.WIG-4.320.9 (deposited with theATCC as accession no. 209534), with or without any signal sequence, andwith or without an N-terminal methionine.

The term “clone 65 variant” means an active clone 65 polypeptide asdefined below having at least about 80%, preferably at least about 85%,more preferably at least about 90%, most preferably at least about 95%amino acid sequence identity with human clone 65 having the deducedamino acid sequence shown in FIGS. 5A and 5B (SEQ ID NO:3) and/or withmouse clone 65 having the deduced amino acid sequence shown in FIGS. 1Aand 1B (SEQ ID NO:6). Such variants include, for instance, clone 65polypeptides wherein one or more amino acid residues are added to, ordeleted from, the N- or C-terminus of the full-length sequences of FIGS.5A-B and 1A-B (SEQ ID NOS:3 and 6, respectively), including variantsfrom other species, but excludes a native-sequence clone 65 polypeptide.

The term “clone 320 variant” means an active clone 320 polypeptide asdefined below having at least about 80%, preferably at least about 85%,more preferably at least about 90%, most preferably at least about 95%amino acid sequence identity with mouse clone 320 derived from the clonedeposited with the ATCC under ATCC no. 209534. Such variants include,for instance, clone 320 polypeptides wherein one or more amino acidresidues are added to, or deleted from, the N- or C-terminus of thesequence encoding mouse clone 320 contained within the about 3-kb insertof pRK5E.m.WIG-4.320.9 deposited with the ATCC under accession no.209534, including variants from other species, but excludes anative-sequence clone 320 polypeptide.

“Percent (%) amino acid sequence identity” with respect to the clone 65and 320 sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a clone 65 or 320 polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,ALIGN, or Megalign (DNASTAR) software. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared.

“Percent (%) nucleic acid sequence identity” with respect to the codingregion of the clone 65 and 320 sequences identified herein is defined asthe percentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the coding region of the clone 65 or clone 320sequence of interest, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, ALIGN, or Megalign (DNASTAR™) software. Those skilled in theart can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared.

“Stringent conditions” are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; (3) employ50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS; or (4) employa buffer of 10% dextran sulfate, 2×SSC (sodium chloride/sodium citrate),and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing LDTA at 55° C.

“Moderately stringent conditions” are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), and include the use of a washing solution andhybridization conditions (e.g., temperature, ionic strength, and percentSDS) less stringent than described above. An example of moderatelystringent conditions is a condition such as overnight incubation at 37°C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc., as necessary to accommodate factors such as probe lengthand the like.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the naturalenvironment of clone 65 or 320 polypeptide will not be present.Ordinarily, however, isolated polypeptide will be prepared by at leastone purification step.

An “isolated” nucleic acid encoding a clone 65 or 320 polypeptide is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the respective nucleic acid. An isolated clone65-encoding or clone 320-encoding nucleic acid molecule is other than inthe form or setting in which it is found in nature. An isolated clone65-encoding or clone 320-encoding nucleic acid molecule therefore isdistinguished from the clone 65-encoding or clone 320-encoding nucleicacid molecule as it exists in natural cells. However, an isolated clone65-encoding or clone 320-encoding nucleic acid molecule includes anucleic acid molecule contained in cells that ordinarily express clone65-encoding and clone 320-encoding DNA, where, for example, the nucleicacid molecule is in a chromosomal location different from that ofnatural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably-linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers single anti-clone 65 or anti-clone 320 monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies), andanti-clone 65 or anti-clone 320 antibodies, and antibody compositionswith polyepitopic specificity. The term “monoclonal antibody” as usedherein refers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts.

“Active” or “activity” or “biological activity”, for purposes herein,describes the activity of form(s) of a clone 65 or 320 polypeptide,including its variants, or its antagonists, which retain the biologicand/or immunologic activities of a native or naturally-occurring(native-sequence) clone 65 or 320 polypeptide or its antagonist.Preferred “activities” for a clone 65 or 320 polypeptide or itsantagonist include the ability to inhibit proliferation of tumor cellsor to stimulate proliferation of normal cells and to treatarteriosclerosis, including atherosclerosis, as well as to induce woundrepair and hematopoiesis, prevent desmoplasia, prevent fibrotic lesionsassociated with skin disorders such as scleroderma, keloid, eosinophilicfasciitus, nodular fasciitis, and Dupuytren's contracture, to treatbone-related diseases such as osteoporosis, to regulate anabolismincluding promotion of growth, to treat immune disorders, to treatWilms' tumor and kidney-related disorders, to treat testis-relateddisorders, to treat lung-related disorders, and to treat cardiacdisorders.

An “antagonist” of a clone 65 or 320 polypeptide is a molecule thatinhibits an activity of a clone 65 or 320 polypeptide. Preferredantagonists are those which interfere with or block an undesirablebiological activity of a clone 65 or 320 polypeptide, such as where aclone 65 or 320 polypeptide might act to stimulate cancer cells and theantagonist would serve to inhibit the growth of those cells. Suchmolecules include antibodies and small molecules that have suchinhibitory capability, as well as polypeptide variants of, and receptorsfor, a clone 65 or 320 polypeptide (if available) or portions thereofthat bind to clone 65 or 320 polypeptide. Thus, the receptor can beexpression cloned from the family; then a soluble form of the receptoris made by identifying the extracellular domain and excising thetransmembrane domain therefrom. The soluble form of the receptor canthen be used as an antagonist, or the receptor can be used to screen forsmall molecules that would antagonize clone 65 or 320 polypeptideactivity.

Alternatively, using the murine nucleotide sequences shown in FIGS. 1-4(SEQ ID NOS:4, 7, 8, or 9, respectively), or the murine amino acidsequence shown in FIG. 1 (SEQ ID NO:6) or the human nucleotide and aminoacid sequences shown in FIGS. 5A-5B (SEQ ID NOS: 1 and 3), variants ofnative clone 65 or clone 320 are made that act as antagonists.

Antagonist activity can be determined by several means, includingstandard assays for induction of cell death such as that describedherein, e.g., ³H-thymidine proliferation assays, or other mitogenicassays, such as an assay measuring the capability of the candidateantagonist of inducing EGF-potentiated anchorage independent growth oftarget cell lines (Volckaert et al., Gene, 15:215-223 (1981)) and/orgrowth inhibition of neoplastic cell lines. Roberts et al., Proc. Natl.Acad. Sci. USA, 82:119-123 (1985). Anchorage-independent growth refersto the ability of clone 65 polypeptide-treated or clone 320polypeptide-treated, or TGF-β-treated and EGF-treated non-neoplastictarget cells to form colonies in soft agar, a characteristic ascribed totransformation of the cells. In this assay, the candidate is incubatedtogether with an equimolar amount of a clone 65 or 320 polypeptideotherwise detectable in the EGF-potentiated anchorage-independent targetcell growth assay, and the culture observed for failure to induceanchorage-independent growth.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder or condition as well as those in which the disorder orcondition is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic, and farm animals, and zoo, sports,or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc.Preferably, the mammal is human.

A “clone 65-related or clone 320-related disorder” is any condition thatwould benefit from treatment with the clone 65 or 320 polypeptides orclone 65 or 320 antagonists herein. This includes chronic and acutedisorders, as well as those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include benign and malignant tumors; leukemias andlymphoid malignancies; neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal, and blastocoelicdisorders; hematopoiesis-related disorders; tissue-growth disorders;skin disorders; desmoplasia, fibrotic lesions; kidney disorders;bone-related disorders; trauma such as burns, incisions, and otherwounds; catabolic states; testicular-related disorders; andinflammatory, angiogenic, and immunologic disorders, includingarteriosclerosis. A “Wnt-related disorder” is one caused at least by theupregulation of the Wnt gene pathway, including Wnt-1 and Wnt-4, butpreferably Wnt-1, and may include cancer.

The terms “cancer”, “cancerous”, and “malignant” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include but are notlimited to, carcinoma including adenocarcinoma, lymphoma, blastoma,melanoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin'slymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer such as hepatic carcinoma and hepatoma, bladdercancer, breast cancer, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer such as renal cellcarcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, and various types of head and neck cancer. The preferred cancersfor treatment herein are breast, colon, lung, and melanoma.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. ¹³¹I,¹²⁵I, ⁹⁰Y, and ¹⁸⁶Re), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant, or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeAdriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere,Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide,Ifosfainide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine,Carboplatin, Teniposide, Daunomycin, Caminomycin, Aminopterin,Dactinomycin, Mitomycins, Esperainicins (see U.S. Pat. No. 4,675,187),Melphalan, and other related nitrogen mustards. Also included in thisdefinition are hormonal agents that act to regulate or inhibit hormoneaction on tumors, such as tamoxifen and onapristone.

A “growth-inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, such as anWnt-overexpressing cancer cell, either in vitro or in vivo Thus, thegrowth-inhibitory agent is one which significantly reduces thepercentage of malignant cells in S phase. Examples of growth-inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxol, and topo II inhibitors such as doxorubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 alsospill over into S-phase arrest, for example, DNA alkylating agents suchas tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in The Molecular Basis of Cancer, Mendelsohn and Israel, eds.,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W B Saunders: Philadelphia,1995), especially p. 13. The 4D5 antibody (and functional equivalentsthereof) can also be employed for this purpose if the cancer involvesErbB2-overexpressing cancer cells. See, e.g., WO 92/22653.

“Northern analysis” or “Northern blot” is a method used to identify RNAsequences that hybridize to a known probe such as an oligonucleotide,DNA fragment, cDNA or fragment thereof, or RNA fragment. The probe islabeled with a radioisotope such as ³²P, or by biotinylation, or with anenzyme. The RNA to be analyzed is usually electrophoretically separatedon an agarose or polyacrylamide gel, transferred to nitrocellulose,nylon, or other suitable membrane, and hybridized with the probe, usingstandard techniques well known in the art such as those described insections 7.39-7.52 of Sambrook et al., supra.

The technique of “polymerase chain reaction,” or “PCR,” as used hereingenerally refers to a procedure wherein minute amounts of a specificpiece of nucleic acid, RNA and/or DNA, are amplified as described inU.S. Pat. No. 4,683,195 issued 28 Jul. 1987. Generally, sequenceinformation from the ends of the region of interest or beyond needs tobe available, such that oligonucleotide primers can be designed; theseprimers will be identical or similar in sequence to opposite strands ofthe template to be amplified. The 5′ terminal nucleotides of the twoprimers may coincide with the ends of the amplified material. PCR can beused to amplify specific RNA sequences, specific DNA sequences fromtotal genomic DNA, and cDNA transcribed from total cellular RNA,bacteriophage, or plasmid sequences, etc. See generally Mullis et al.,Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCRTechnology, (Stockton Press, NY, 1989). As used herein, PCR isconsidered to be one, but not the only, example of a nucleic acidpolymerase reaction method for amplifying a nucleic acid test samplecomprising the use of a known nucleic acid as a primer and a nucleicacid polymerase to amplify or generate a specific piece of nucleic acid.

II. Compositions and Methods of the Invention

A. Full-length Clone 65 or 320 Polypeptide

The present invention provides newly identified and isolated nucleotidesequences encoding a polypeptide referred to in the present applicationas a clone 65 or clone 320 polypeptide. In particular, cDNAs have beenidentified and isolated encoding novel murine and human clone 65polypeptides as well as murine clone 320 as disclosed in further detailin the Examples below.

Using BLAST and FastA sequence alignment computer programs, it was foundthat the coding sequences of mouse and human clone 65, as well as thethree sequences for mouse clone 320 disclosed herein, show significanthomology to DNA sequences disclosed in the GenBank database, includingthose published by Adams et al., Nature, 377: 3-174 (1995).

Using BLAST and FastA sequence alignment computer programs, it was foundthat mouse and human clone 65 show significant homology to members ofthe Rho family of small GTPases (mouse and human clone 65 are 58-59% and59% homologous, respectively, to human G25k gtp-binding protein,placental and brain isoforms, and 59% homologous to canine CDC42GTP-binding protein). Accordingly, it is presently believed that theclone 65 polypeptides disclosed in the present application possessactivity relating to the treatment of various cancers with which Ras isassociated, as well as to arteriosclerosis, such as atherosclerosis.

B. Clone 65 and 320 Polypeptide Variants

In addition to the full-length native-sequence clone 65 and 320polypeptides described herein, it is contemplated that variants of thesesequences can be prepared. Clone 65 and 320 variants can be prepared byintroducing appropriate nucleotide changes into the clone 65-encodingand clone 320-encoding DNA, or by synthesis of the desired variant clone65 and 320 polypeptides. Those skilled in the art will appreciate thatamino acid changes may alter post-translational processes of the clone65 and 320 polypeptides, such as changing the number or position ofglycosylation sites or altering the membrane-anchoring characteristics,if the native clone 65 or 320 polypeptide is membrane bound.

Variations in the native full-length clone 65 and 320 sequences, or invarious domains of the clone 65 and 320 polypeptides described herein,can be made, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations may be a substitution, deletion, orinsertion of one or more codons encoding the clone 65 and 320polypeptide that results in a change in the amino acid sequence ascompared with the native-sequence clone 65 and 320 polypeptide.Optionally the variation is by substitution of at least one amino acidwith any other amino acid in any portion of the clone 65 and 320polypeptide. Guidance in determining which amino acid residue may beinserted, substituted, or deleted without adversely affecting thedesired activity may be found by comparing the sequence of the clone 65and 320 polypeptide with that of homologous known Rae or Rho proteinmolecules, in the case of clone 65, and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of 1 to about 5 amino acids. The variation allowed may bedetermined by systematically making insertions, deletions, orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity in in vitro assays for gene upregulation ordownregulation and in transgenic or knock-out animals.

The variations can be made on the cloned DNA to produce variant DNAusing methods known in the art such as oligonucleotide-mediated(site-directed) mutagenesis (Carter et al., Nucl. Acids Res, 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), alanine scanning, PCRmutagenesis, restriction selection mutagenesis (Wells et al., Philos.Trans. R. Soc. London SerA, 317:415 (1986)), or other known techniques.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions. T. E.Creighton, Proteins: Structure and Molecular Properties (W.H. Freeman &Co., San Francisco, 1983); Chothia, J. Mol. Biol., 150:1 (1976). Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Further deletional variants of the full-length clone 65 and 320polypeptides include variants from which the N-terminal signal peptide,if any, and/or the initiating methionine has been deleted.

C. Modifications of the Clone 65 and 320 Polypeptides

Covalent modifications of the clone 65 and 320 polypeptides are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of a clone 65 or 320polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues.Derivatization with bifunctional agents is useful, for instance, forcrosslinking a clone 65 or 320 polypeptide to a water-insoluble supportmatrix or surface for use in the method for purifying anti-clone 65antibodies and anti-clone 320 antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane, and agentssuch as methyl-3-((p-azidophenyl)dithio)pro-pioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains(Creighton, supra, pp. 79-86), acetylation of the N-terminal amine, andamidation of any C-terminal carboxyl group.

Another type of covalent modification of the clone 65 or 320 polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in the native sequence (eitherby deleting the underlying glycosylation site or by removing theglycosylation moieties by chemical and/or enzymatic means) and/or addingone or more glycosylation sites that are not present in the nativesequence. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportion of the various sugar residues present.

Addition of glycosylation sites to the clone 65 or 320 polypeptideherein may be accomplished by altering the amino acid sequence. Thealteration may be made, for example, by the addition of, or substitutionby, one or more serine or threonine residues to the native sequence (forO-linked glycosylation sites). The amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the clone 65 or 320 polypeptide at preselected bases suchthat codons are generated that will translate into the desired aminoacids. The DNA mutation(s) may be made using methods described above.

Another means of increasing the number of carbohydrate moieties on theclone 65 or 320 polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the clone 65 or 320polypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biopyhs., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification comprises linking the clone 65 or320 polypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth, e.g., in U.S. Pat. No. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The clone 65 or 320 polypeptide of the present invention may also bemodified in a way to form a chimeric molecule comprising a clone 65 or320 polypeptide, or a fragment thereof, fused to a heterologouspolypeptide or amino acid sequence. In one embodiment, such a chimericmolecule comprises a fusion of the clone 65 or 320 polypeptide with atag polypeptide which provides an epitope to which an anti-tag antibodycan selectively bind. The epitope tag is generally placed at the amino-or carboxyl-terminus of a native or variant clone 65 or 320 molecule.The presence of such epitope-tagged forms can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the clone 65 or 320 polypeptide to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of the clone 65or 320 polypeptide, or fragments thereof, with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an Ig,such as an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7, and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody. Paborsky et al, Protein Engineering, 3(6):547-553(1990). Other tag polypeptides include the Flag-peptide (Hopp et al.,BioTechnology, 6:1204-1210 (1988)); the KT3 epitope peptide (Martin etal., Science, 255:192-194 (1992)); an α-tubulin epitope peptide (Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)); and the T7 gene 10protein peptide tag. Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990).

D. Preparation of Clone 65 and 320 Polypeptides

The description below relates primarily to production of clone 65 and320 polypeptides by culturing cells transformed or transfected with avector containing at least DNA encoding the mature or full-lengthsequences of human or mouse clone 65 (SEQ ID NOS:3 or 6, respectively),or containing at least mouse clone 320 DNA (deposited at the ATCC asaccession no. 209534 and comprising SEQ ID NOS:7, 8, and/or 9).

It is, of course, contemplated that alternative methods, which are wellknown in the art, may be employed to prepare clone 65 and 320polypeptides. For instance, the clone 65 or 320 polypeptide sequence, orportions thereof, may be produced by direct peptide synthesis usingsolid-phase techniques. See, e.g., Stewart et al., Solid-Phase PeptideSynthesis (W.H. Freeman Co.: San Francisco, Calif., 1969); Merrifield,J. Am. Chem. Soc., 85:2149-2154 (1963). In vitro protein synthesis maybe performed using manual techniques or by automation. Automatedsynthesis may be accomplished, for instance, using an Applied Biosystemspeptide synthesizer (Foster City, Calif.) in accordance withmanufacturer's instructions. Various portions of clone 65 and 320polypeptides may be chemically synthesized separately and combined usingchemical or enzymatic methods to produce a full-length clone 65 or 320polypeptide.

1. Isolation of DNA Encoding Clone 65 and 320 Polypeptides

DNA encoding a clone 65 or 320 polypeptide may be obtained from a cDNAlibrary prepared from tissue believed to possess the mRNA for clone 65or 320 polypeptide and to express it at a detectable level. Accordingly,DNA encoding human clone 65 or 320 polypeptide can be convenientlyobtained from a cDNA library prepared from human tissue, such as a humanfetal liver library or as otherwise described in the Examples. The genesencoding clone 65 and 320 polypeptides may also be obtained from agenomic library or by oligonucleotide synthesis.

A still alternative method of cloning clone 65 or 320 polypeptide issuppressive subtractive hybridization, which is a method for generatingdifferentially regulated or tissue-specific cDNA probes and libraries.This is described, for example, in Diatchenko et al., Proc. Natl. Acad.Sci. USA, 93: 6025-6030 (1996). The procedure is based primarily on atechnique called suppression PCR and combines normalization andsubtraction in a single procedure. The normalization step equalizes theabundance of cDNAs within the target population and the subtraction stepexcludes the common sequences between the target and driver populations.

Libraries can be screened with probes (such as antibodies to a clone 65or 320 polypeptide or oligonucleotides of at least about 20-80 bases)designed to identify the gene of interest or the protein encoded by it.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures, such as described in Sambrook etal., supra. An alternative means to isolate the gene encoding clone 65or 320 polypeptide is to use PCR methodology. Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1995).

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation, or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as ALIGN, DNAstar, and INHERIT which employ various algorithms tomeasure homology.

Nucleic acid having polypeptide-coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequences disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for clone 65 or 320 polypeptide production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH, and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: a Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene or polyornithine, may also beused. For various techniques for transforming mammalian cells, see Keownet al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceac such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC53,635). These examples are illustrative rather than limiting. StrainW3110 is one particularly preferred host or parent host because it is acommon host strain for recombinant DNA product fermentations.Preferably, the host cell secretes minimal amounts of proteolyticenzymes. For example, strain W3110 may be modified to effect a geneticmutation in the genes encoding proteins endogenous to the host, withexamples of such hosts including E. coli W3110 strain 1A2, which has thecomplete genotype tonA; E. coli W310 strain 9E4, which has the completegenotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan^(r); E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan^(r) ; E. coli W3110strain 40B4, which is strain 37D6 with a non-kanamycin resistant degPdeletion mutation; and an E. coli strain having mutant periplasmicprotease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990.Alternatively, in vitro methods of cloning, e.g., PCR or other nucleicacid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for vectorscontaining nucleic acid encoding clone 65 or 320 polypeptide.Saccharomyces cerevisiae is a commonly used lower eukaryotic hostmicroorganism. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe (Beach and Nurse, Nature, 290: 140 (1981); EP 139,383 published 2May 1985); Kluyverornyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9: 968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 (1983)), K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van denBerg et al., Bio/Technology, 8: 135 (1990)), K. thermotolerans, and K.marxianus, yarrowia (EP 402,226); Pichia pastoris (EP 183,070;Sreekrishna et al., J. Basic Microbiol., 28: 265-278 (1988)); Candida,Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc.Natl. Acad. Sci. USA, 76: 5259-5263 (1979)); Schwanniomyces such asSchwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A.nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289(1983); Tilburn et al., Gene, 26: 205-221 (1983); Yelton et al., Proc.Natl. Acad. Sci. USA, 81: 1470-1474 (1984)) and A. niger Kelly andHynes, EMBO J., 4: 475-479 (1985). Methylotropic yeasts are suitableherein and include, but are not limited to, yeast capable of growth onmethanol selected from the genera consisting of Hansenula, Candida,Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list ofspecific species that are exemplary of this class of yeasts may be foundin C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated clone 65 or 320polypeptide are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture (Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells(TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammarytumor (MMT 060562, ATCC CCL51). The selection of the appropriate hostcell is deemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the desired clone65 or 320 polypeptide may be inserted into a replicable vector forcloning (amplification of the DNA) or for expression. Various vectorsare publicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The desired clone 65 or 320 polypeptide may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence, if the clone 65 or 320polypeptide is conducive to being secreted, or other polypeptide havinga specific cleavage site at the N-terminus of the mature or full-lengthprotein or polypeptide. In general, the signal sequence may be acomponent of the vector, or it may be a part of the DNA encoding theclone 65 or 320 polypeptide that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence such as, for example, thealkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin IIleaders. For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces a-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders, and including signals fromclone 65 or 320 polypeptide.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV, or BPV) areuseful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g. the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the nucleicacid encoding clone 65 or 320 polypeptide, such as DHFR or thymidinekinase. An appropriate host cell when wild-type DHFR is employed is theCHO cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7. Stinchcomb et al. Nature, 282:39 (1979);Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157(1980). The irp1 gene provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example, ATCC No.44076 or PEP4-1. Jones, Genetics, 85:12 (1977).

Expression and cloning vectors usually contain a promoter operablylinked to the nucleic acid sequence encoding clone 65 or 320 polypeptideto direct mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems (Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)),alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776), and hybrid promoters suchas the tac promoter. deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983). Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theclone 65 or 320 polypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

Clone 65 or 320 transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus,and Simian Virus 40 (SV40); from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter; and from heat-shockpronoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding a clone 65 or 320 polypeptide by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thesequence coding for a clone 65 or 320 polypeptide, but is preferablylocated at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding clone 65 or 320 polypeptide.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of clone 65 and 320 polypeptides in recombinant vertebratecell culture are described in Gething et al., Nature, 293:620-625(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native-sequenceclone 65 or 320 polypeptide or against a synthetic peptide based on theDNA sequences provided herein or against exogenous sequence fused to DNAencoding clone 65 or 320 polypeptide and encoding a specific antibodyepitope.

5. Purification of Polypeptide

Forms of clone 65 or 320 polypeptide may be recovered from culturemedium or from host cell lysates. If membrane-bound, it can be releasedfrom the membrane using a suitable detergent solution (e.g., Triton-X100) or by enzymatic cleavage. Cells employed in expression of clone 65and 320 polypeptides can be disrupted by various physical or chemicalmeans, such as freeze-thaw cycling, sonication, mechanical disruption,or cell lysing agents.

It may be desired to purify clone 65 or 320 polypeptide from recombinantcell proteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, SEPHADEX™ G-75; protein A SEPHAROSE™ columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the clone 65 or 320 polypeptide. Various methodsof protein purification may be employed, and such methods are known inthe art and described, for example, in Deutscher, Methods in Enzymology,182 (1990); and Scopes, Protein Purification:Principles and Practice(Springer-Verlag: New York, 1982).

In one specific example of purification, either a poly-HIS tag or the Fcportion of human IgG is added to the C-terminal coding region of thecDNA for clone 65 or clone 320 before expression. The conditioned mediafrom the transfected cells are harvested by centrifugation to remove thecells and filtered. For the poly-HIS tagged constructs, the protein maybe purified using a Ni-NTA column. After loading, the column may bewashed with additional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein may then be desalted into a storage buffer if desired.

Immunoadhesin (Fc-containing) constructs of the clone 65 or clone 320polypeptides may be purified from the conditioned media by pumping themonto a 5-ml Protein A column that had been equilibrated in a phosphatebuffer. After loading, the column may be washed extensively withequilibration buffer before elution with citric acid. The eluted proteinmay be immediately neutralized by collecting 1-ml fractions into tubescontaining TRIS buffer. The highly purified protein may be subsequentlydesalted into storage buffer as described above for the poly-HIS taggedproteins. The homogeneity of the protein may be assessed by SDSpolyacrylamide gels and by N-terminal amino acid sequencing by Edmandegradation.

The purification step(s) selected will depend, for example, on thenature of the production process used and the particular clone 65 or 320polypeptide produced.

E. Uses for Clone 65 and 320 Polypeptides and Their Nucleic Acid

Nucleotide sequences (or their complement) encoding clone 65 and 320polypeptides have various applications in the art of molecular biology,including uses as hybridization probes, in chromosome and gene mapping,and in the generation of anti-sense RNA and DNA. Nucleic acid encodingclone 65 or 320 polypeptide will also be useful for the preparation ofclone 65 or 320 polypeptide by the recombinant techniques describedherein.

The full-length nucleotide sequences for mouse or human clone 65 (SEQ IDNOS:4 and 1, respectively), or portions thereof, or the sequences inFIGS. 2, 3, and 4 for mouse clone 320 (SEQ ID NOS:7, 8, and/or 9), orportions thereof, may be used as hybridization probes for a cDNA libraryto isolate or detect the full-length gene encoding the clone 65 or 320polypeptide of interest or to isolate or detect still other genes (forinstance, those encoding naturally occurring variants of clone 65 or 320polypeptide, other clone 65 or 320 polypeptide family members, or clone65 or 320 polypeptides from other species) which have a desired sequenceidentity to the clone 65 polypeptide sequences disclosed in FIGS. 1 and5 (SEQ ID NOS:6 and 3, respectively). For example, such procedures as insitu hybridization, Northern and Southern blotting, and PCR analysis maybe used to determine whether DNA and/or RNA encoding a different clone65 or 320 polypeptide is present in the cell type(s) being evaluated.Optionally, the length of the probes will be about 20 to about 50 bases.For example, the hybridization probes may be derived from ESTs, clonedsequences, or genomic sequences including promoters, enhancer elements,and introns of DNA encoding native-sequence clone 65 or 320 polypeptide.

By way of example, a screening method will comprise isolating the codingregion of the clone 65 or clone 320 gene using the known DNA sequence tosynthesize a selected probe of about 40 bases. Hybridization probes maybe labeled by a variety of labels, including radionucleotides such as³²P or ³⁵S, or enzymatic labels such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems. Labeled probes having asequence complementary to that of any of the genes encoding clone 65 or320 polypeptide of the present invention can be used to screen librariesof human cDNA, genomic DNA, or mRNA to determine to which members ofsuch libraries the probe hybridizes. Hybridization techniques aredescribed in further detail in the Examples below.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely-related clone 65 and 320sequences. Nucleotide sequences encoding a clone 65 or 320 polypeptidecan also be used to construct hybridization probes for mapping the genethat encodes the particular clone 65 or 320 polypeptide and for thegenetic analysis of individuals with genetic disorders. The nucleotidesequences provided herein may be mapped to a chromosome and specificregions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

Nucleic acid encoding a clone 65 or 320 polypeptide may be used as adiagnostic to determine the extent and rate of the expression of the DNAencoding a clone 65 or 320 polypeptide in the cells of a patient. Toaccomplish such an assay, a sample of a patient's cells is treated, viain situ hybridization, or by other suitable means, and analyzed todetermine whether the sample contains mRNA molecules capable ofhybridizing with the nucleic acid molecule.

Nucleic acids that encode clone 65 or 320 polypeptide or any of theirmodified forms can also be used to generate either transgenic animals or“knock-out” animals which, in turn, are useful in the development andscreening of therapeutically useful reagents. A transgenic animal (e.g.,a mouse or rat) is an animal having cells that contain a transgene,which transgene was introduced into the animal or an ancestor of theanimal at a prenatal, e.g. an embryonic stage. A transgene is a DNA thatis integrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding a clone 65 or 320 polypeptidecan be used to clone genomic DNA encoding the clone 65 or 320polypeptide in accordance with established techniques and the genomicsequences used to generate transgenic animals that contain cells whichexpress DNA encoding the clone 65 or 320 polypeptide.

Methods for generating transgenic animals, particularly animals such asmice or rats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009 and WO 97/38086.Typically, particular cells would be targeted for clone 65 or 320transgene incorporation with tissue-specific enhancers. Transgenicanimals that include a copy of a transgene encoding the clone 65 or 320polypeptide introduced into the germ line of the animal at an embryonicstage can be used to examine the effect of increased expression of DNAencoding the clone 65 or 320 polypeptide. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of clone 65 and 320 polypeptides canbe used to construct a clone 65 or 320 polypeptide “knock-out” animalwhich has a defective or altered gene encoding a clone 65 or 320polypeptide as a result of homologous recombination between theendogenous gene encoding the clone 65 or 320 polypeptide and alteredgenomic DNA encoding the clone 65 or 320 polypeptide introduced into anembryonic cell of the animal. For example, cDNA encoding the clone 65 or320 polypeptide can be used to clone genomic DNA encoding the clone 65or 320 polypeptide in accordance with established techniques. A portionof the genomic DNA encoding the clone 65 or 320 polypeptide can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector. See e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors. The vectoris introduced into an embryonic stem cell line (e.g. by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected. See e.g., Li et al., Cell, 69:915(1992). The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras. See e.g.Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a“knock-out” animal. Progeny harboring the homologously recombined DNA intheir germ cells can be identified by standard techniques and used tobreed animals in which all cells of the animal contain the homologouslyrecombined DNA. Knock-out animals can be characterized, for instance, bytheir ability to defend against certain pathological conditions and bytheir development of pathological conditions due to absence of the clone65 or 320 polypeptide.

In particular, assays in which the Rac and Rho family members areusually used are preferably performed with the clone 65 polypeptide.Further, an assay to determine whether TGF-β induces the clone 65 or 320polypeptide, indicating a role in cancer, may be performed as known inthe art, as well as assays involving induction of cell death and3H-thymidine proliferation assays. Mitogenic and tissue growth assaysare also performed with the clone 65 or 320 polypeptide as set forthabove. The results are applied accordingly.

The clone 65 or 320 polypeptides of the present invention may also beused to induce the formation of anti-clone 65 or anti-clone 320polypeptide antibodies, which are identified by routine screening asdetailed below.

For diagnostic purposes, the clone 65 or clone 320 polypeptide can beused in accordance with immunoassay technology. Examples of immunoassaysare provided by Wide at pages 199-206 of Radioimmune Assay Method,Kirkham and Huner, ed., E & S. Livingstone, Edinburgh, 1970.

Thus, in one embodiment, clone 65 or clone 320 polypeptides can bedetectably labeled and incubated with a test sample containing themolecules of interest (such as biological fluids, e.g., serum, sputum,urine, etc.), and the amount of clone 65 or clone 320 molecule bound tothe sample ascertained.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the clone 65 or clone 320 polypeptidefrom any analyte that remains free in solution. This conventionally isaccomplished by either insolubilizing the clone 65 or clone 320polypeptide before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the molecule afterward, e.g., byimmunoprecipitation.

The foregoing are merely exemplary diagnostic assays. Other methods nowor hereafter developed for the determination of these analytes areincluded within the scope hereof.

In addition, clone 65 and 320 polypeptides are useful for screening forcompounds that bind to them as defined above. Preferably, thesecompounds are small molecules such as organic or peptide molecules thatexhibit one or more of the desired activities. Screening assays of thiskind are conventional in the art, and any such screening procedure maybe employed, whereby the test sample is contacted with the clone 65 or320 polypeptide herein and the extent of binding and biological activityof the bound molecule are determined.

Clone 65 and clone 320 polypeptides are additionally useful in affinitypurification of a molecule that binds to clone 65 or clone 320polypeptide and in purifying antibodies thereto. The clone 65 or clone320 polypeptide is typically coupled to an immobilized resin such asAffi-Gel 10™ (Bio-Rad, Richmond, Calif.) or other such resins (supportmatrices) by means well known in the art. The resin is equilibrated in abuffer (such as one containing 150 mM NaCl, 20 mM HEPES, pH 7.4supplemented to contain 20% glycerol and 0.5% NP-40) and the preparationto be purified is placed in contact with the resin, whereby themolecules are selectively adsorbed to the clone 65 or clone 320polypeptide on the resin.

The resin is then sequentially washed with suitable buffers to removenon-adsorbed material, including unwanted contaminants, from the mixtureto be purified, using, e.g., 150 mM NaCl, 20 mM HEPES, pH 7.4,containing 0.5% NP-40; 150 mM NaCl, 20 mM HEPES, pH 7.4 containing 0.5 MNaCl and 0.1% NP-40; 150 mM NaCl, 20 mM HEPES, pH 7.4 containing 0.1%deoxycholate; 150 mM NaCl, 20 mM HEPES, pH 7.4 containing 0.1% NP-40;and a solution of 0.1% NP-40, 20% glycerol and 50 mM glycine, pH 3. Theresin is then treated so as to elute the binding molecule using a bufferthat will break the bond between the binding molecule and clone 65 orclone 320 polypeptide (using, e.g., 50 mM glycine, pH 3, 0.1% NP-40, 20%glycerol, and 100 mM NaCl).

It is contemplated that the clone 65 and 320 polypeptides of the presentinvention may be used to treat various conditions, including thosecharacterized by overexpression and/or activation of at least the Wntpathway. Further, they are useful in diagnosing cancer, for example, asa marker for increased susceptibility to cancer or for having cancer.Exemplary conditions or disorders to be treated with the clone 65 and320 polypeptides include benign or malignant tumors (e.g., renal, liver,kidney, bladder, testicular, breast, gastric, ovarian, colorectal,prostate, pancreatic, lung, esophageal, vulval, thyroid, hepaticcarcinomas; sarcomas; glioblastomas; and various head and neck tumors);leukemias and lymphoid malignancies; other disorders such as neuronal,glial, astrocytal, hypothalamic, and other glandular, macrophagal,epithelial, stromal, and blastocoelic disorders; cardiac disorders;renal disorders; catabolic disorders; bone-related disorders such asosteoporosis; and inflammatory, angiogenic, and immunologic disorders,such as arteriosclerosis.

The clone 65 and 320 polypeptides of the invention are administered to amammal, preferably a human, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the polypeptide is preferred.

Therapeutic formulations of the clone 65 or 320 polypeptide are preparedfor storage by mixing the polypeptide having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients,or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose, or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

Other therapeutic regimens may be combined with the administration ofthe clone 65 and 320 polypeptides of the instant invention. For example,the patient to be treated with the polypeptides disclosed herein mayalso receive radiation therapy if the disorder is cancer. Alternatively,or in addition, a chemotherapeutic agent may be administered to thepatient with cancer. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service, Ed., M. C. Perry (Williams & Wilkins:Baltimore, Md., 1992). The chemotherapeutic agent may precede or followadministration of the polypeptide or may be given simultaneouslytherewith. The polypeptide may be combined with an anti-oestrogencompound such as tamoxifen or an anti-progesterone such as onapristone(see, EP 616812) in dosages known for such molecules.

It may be desirable also to co-administer with the clone 65 or 320polypeptide (or anti-clone 65 or anti-clone 320 polypeptide antibodies)antibodies against other tumor-associated antigens, such as antibodieswhich bind to HER-2, EGFR, ErbB2, ErbB3, ErbB4, or vascular endothelialfactor (VEGF). Alternatively, or in addition, two or more differentanti-cancer antibodies, such as anti-ErbB2 antibodies, may beco-administered to the patient with the clone 65 or 320 polypeptide (oranti-clone 65 or anti-clone 320 polypeptide antibodies). Sometimes, itmay be beneficial also to administer one or more cytokines to thepatient.

In a preferred embodiment, the clone 65 or clone 320 polypeptide isco-administered with a growth-inhibitory agent to the cancer patient.For example, the growth-inhibitory agent may be administered first,followed by the clone 65 or 320 polypeptide. However, simultaneousadministration or administration of the clone 65 or clone 320polypeptide first is also contemplated. Suitable dosages for thegrowth-inhibitory agent are those presently used and may be lowered dueto the combined action (synergy) of the growth-inhibitory agent andpolypeptide. The antibodies, cytotoxic agents, cytokines, orgrowth-inhibitory agents are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively,in colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles, and nanocapsules) or inmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980), supra. Theformulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the polypeptide, which matrices are inthe form of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated polypeptides remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

For the prevention or treatment of disease or disorder, the appropriatedosage of clone 65 or 320 polypeptide will depend on the type ofdisorder to be treated, as defined above, the severity and course of thedisorder, whether the polypeptide is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the polypeptide, the route of administration, thecondition of the patient, and the discretion of the attending physician.The polypeptide is suitably administered to the patient at one time orover a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1-20 mg/kg) of clone 65 or 320 polypeptide is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of symptoms ofthe disorder occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example, the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the clone 65 or 320polypeptide. The label on, or associated with, the container indicatesthat the composition is used for treating the condition or disorder ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asphosphate-buffered saline, Ringer's solution, and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

F. Anti-Clone 65 and 320 Polypeptide Antibodies

The present invention further provides anti-clone 65 and 320 polypeptideantibodies. Exemplary antibodies include polyclonal, monoclonal,humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-clone 65 and 320 polypeptide antibodies of the presentinvention may comprise polyclonal antibodies. Methods of preparingpolyclonal antibodies are known to the skilled artisan. Polyclonalantibodies can be raised in a mammal, for example, by one or moreinjections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the clone 65 or 320 polypeptide or a fusionprotein thereof. It may be useful to conjugate the immunizing agent to aprotein known to be immunogenic in the mammal being immunized. Examplesof such immunogenic proteins include but are not limited to keyholelimpet hemocyanin, serum albumin, bovine thyroglobulin, and soybeantrypsin inhibitor. Examples of adjuvants which may be employed includeFreund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-clone 65 or 320 polypeptide antibodies may, alternatively, bemonoclonal antibodies. Monoclonal antibodies may be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, orother appropriate host animal is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the clone 65 or 320polypeptide or a fusion protein thereof. Generally, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such as PEG, toform a hybridoma cell. Goding, Monoclonal Antibodies: Principles andPractice (Academic Press: New York, 1986) pp. 59-103. Immortalized celllines are usually transformed mammalian cells, particularly myelomacells of rodent, bovine, and human origin. Usually, rat or mouse myelomacell lines are employed. The hybridoma cells may be cultured in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, immortalized cells.For example, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high-level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif., and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications (MarcelDekker, Inc.: New York, 1987) pp. 51-63.

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against aclone 65 or 320 polypeptide. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, supra. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, CHO cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy-chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart.

3. Humanized Antibodies

The anti-clone 65 and anti-clone 320 polypeptide antibodies of theinvention may further comprise humanized antibodies or human antibodies.Humanized forms of non-human (e.g. murine) antibodies are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)₂, or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin, and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody preferably also will compriseat least a portion of an immunoglobulin constant region (Fe), typicallythat of a human immunoglobulin. Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op.Struct. Biol., 2:593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al. Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage-display libraries. Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991).The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies. Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner etal., J. Immunol., 147(1):86-95 (1991).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aclone 65 or 320 polypeptide; the other one is for any other antigen, andpreferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities. Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant-domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection. WO 91/00360; WO 92/200373; EP 03089.It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

G. Uses for Anti-Clone 65 and Anti-Clone 320 Polypeptide Antibodies

The antibodies of the invention may be used as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a SEPHADEX™ resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theclone 65 or 320 polypeptide (or fragment thereof) to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the clone 65or 320 polypeptide, which is bound to the immobilized antibody. Finally,the support is washed with another suitable solvent, such as glycinebuffer, pH 5.0, that will release the clone 65 or 320 polypeptide fromthe antibody.

Anti-clone 65 or 320 polypeptide antibodies may also be useful indiagnostic assays for clone 65 or 320 polypeptide, e.g., detecting itsexpression in specific cells, tissues, or serum. Thus, the antibodiesmay be used in the diagnosis of human malignancies (see, for example,U.S. Pat. No. 5,183,884).

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can bepreferably grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.,(Wiley-Interscience: New York, 1991), for example, and radioactivity canbe measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin, and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, Coligen,ed., for example. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available, and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme preferablycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym., Vol. 73, Langone and VanVunakis, eds. (New York: Academic Press, 1981), pp. 147-166.

Examples of enzyme-substrate combinations include:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g. orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate(4-methylumbelliferyl-β-D-galactosidase).

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see, for example,U.S. Pat. Nos. 4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin, and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g, anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the anti-clone 65 or 320polypeptide antibody need not be labeled, and the presence thereof canbe detected using a labeled antibody that binds to the anti-clone 65 or320 polypeptide antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques (New York: CRC Press, Inc., 1987),pp. 147-158.

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of clone 65 or 320 polypeptide in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies preferably are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Preferably, the antibody is labeled with a radionuclide (such as ¹¹¹In,⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

Additionally, anti-clone 65 or 320 polypeptide antibodies may be usefulas antagonists to clone 65 or 320 polypeptide functions where clone 65or 320 polypeptide is upregulated in cancer cells or stimulates theirprolilferation or is upregulated in atherosclerotic tissue. Hence, forexample, the anti-clone 65 and 320 polypeptide antibodies may bythemselves or with a chemotherapeutic agent or other cancer treatment ordrug such as anti-HER-2 antibodies be effective in treating certainforms of cancer such as breast cancer, colon cancer, lung cancer, andmelanoma. Further uses for the antibodies include inhibiting the bindingof a clone 65 or 320 polypeptide to its receptor, if applicable, or to aprotein that binds to the clone 65 or 320 polypeptide, if applicable.For therapeutic use, the antibodies can be used in the formulations,schedules, routes, and doses indicated above under uses for the clone 65and 320 polypeptides. In addition, anti-clone 65 and 320 polypeptideantibodies may be administered into the lymph as well as the bloodstream.

As a matter of convenience, the anti-clone 65 or 320 polypeptideantibody of the present invention can be provided in a kit format, i.e.,a packaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit will include substrates and cofactorsrequired by the enzyme (e.g. a substrate precursor which provides thedetectable chromophore or fluorophore). In addition, other additives maybe included such as stabilizers, buffers (e.g. a block buffer or lysisbuffer), and the like. The relative amounts of the various reagents maybe varied to provide for concentrations in solution of the reagentswhich substantially maximize the sensitivity of the assay. Particularly,the reagents may be provided as dry powders, usually lyophilized,including excipients which on dissolution will provide a reagentsolution having the appropriate concentration.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, 10801 University Blvd., Manassas, Va.

Example 1 Isolation of cDNA Clones Encoding Mouse Clone 65

Several putative genes encoding clone 65 and 320 polypeptides have beenidentified at the mRNA level in a high throughput PCR-select cDNAsubstraction experiment carried out using a mouse mammary cell line(C57MG), which has been transformed by a Wnt-1 retroviral vector andcompared with the parental cell line. The clone 65 and 320 polypeptidefamily disclosed herein, including the mouse clone 65 gene, was inducedonly in the transformed cell line C57MGWnt-1.

1. Suppression Subtractive Hybridization

Mouse clone 65 was isolated independently by Wnt-1 differentialscreening using suppression subtractive hybridization (SSH), asdescribed by Diatchenko et al., Proc. Natl. Acad. Sci. USA, 93:6025-6030 (1996). SSH was carried out using the PCR-SELECT® cDNASubtraction Kit (Clontech Laboratories, Inc.) according to themanufacturer's protocol. Driver double-stranded (ds) cDNA wassynthesized from 2 micrograms of polyA4+ RNA isolated from a mousemammary cell line (C57MG), obtainable from a mouse breast cancermyoepithelial cell line. This cell line is described in Brown et al.,Cell, 46: 1001-1009 (1986); Olson and Papkoff, Cell Growth andDifferentiation, 5: 197-206 (1994); Wong et al., Mol. Cell. Biol., 14:6278-6286 (1994); and Jue et al, Mol. Cell. Biol., 12: 321-328 (1992),and is responsive to Wnt-1 but not to Wnt-4. Tester ds cDNA wassynthesized from 2 micrograms of polyA+ RNA isolated from a transformedversion of C57MG, called C57MG/wnt-1.

The C57MG/wnt-1 mouse mammary derivative cell line was prepared by firsttransforming the parent line with a Wnt-1 retroviral vector, pBabe Puro(5.1 kb). This vector has a 5′ LTR, packaging elements, a multiplecloning site, the puromycin-resistance gene driven off the SV40promoter, a 3′ LTR, and the bacterial elements for replication andampicillin selection. The vector was modified slightly for Wnt-1 cloningby removing the HindIII site after the SV40 promoter and adding aHindIII site to the multiple cloning site. Wnt-1 is cloned fromEcoRI-HindIII in the multiple cloning site. FIG. 7 shows a map of thevector.

The transformed derivative cells were grown up in a conventionalfashion, and the final cell population was selected in DMEM+10% FCS with2.5 μg/ml puromycin to stabilize the expression vector.

PCR was performed using the Clontech kit, including the cDNA synthesisprimer (SEQ ID NO:20), adaptors 1 and 2 (SEQ ID NOS:21 and 22,respectively) and complementary sequences for the adaptors (SEQ IDNOS:23 and 24, respectively), PCR primer 1 (SEQ ID NO:25), PCR primer 2(SEQ ID NO:26), nested PCR primer 1 (SEQ ID NO:27), nested PCR primer 2(SEQ ID NO:28), control primer G3PDH5′ primer (SEQ ID NO:29), andcontrol primer G3PDH3′ primer (SEQ ID NO:30), shown in FIG. 8.

Products generated from the secondary PCR reaction were inserted intothe cloning site region of pGEM-T vector (Promega), shown in FIG. 9 (SEQID NOS:31 and 32 for 5′ and 3′ sequences, respectively). Plasmid DNAswere prepared using the Wizard Miniprep Kit™ (Promega). DNA sequencingof the subcloned PCR fragments was performed manually by the chaintermination reaction (Sequenase 2.0™ Kit, Pharmacia). Nucleic acidhomology searches were performed using the BLAST program noted above.

A total of 1384 clones were sequenced out of greater than 5000 found. Atotal of 1996 DNA templates were prepared. A program was used to trimthe vector off, and a different program used to cluster the clones intotwo or more identical clones or with an overlap of 50 bases the same.Then a BLAST was performed of a representative clone from the cluster.Primers were designed for RT-PCR to see if the clones weredifferentially expressed.

2. Semi-Quantitative RT-PCR

The initial clone isolated, designated clone 65, had 212 bp and thesequence:

(SEQ ID NO:11) 5′CAGAGGGTGGGTGGGAAAGAGTGAATTATTTAATTTTAAATGTTATAATAAAGCCAATGTAGTTGAGACCAAGGAAATGAGCATTGAGAACACAAACTTGAAGTCTGGTGCCAGGGTTGTTGGACCTCACACCCTGTCTCTGAGCCACCCGGAAGTGACATAAAGGACGCTGTGTGATCAAGTTCTGGACACTTTTCT GGGATG.

RT-PCR primers were designed for confirming differential expression,pulling out additional clones, screening for full-length mouse clone,and screening for the human clone. The RT-PCR primers were designed asfollows:

(SEQ ID NO:33) 65.pcr.top1: 5′-CAGAGGGTGGGTGGGAAAGAGTGA and (SEQ IDNO:34) 65.pcr.bot2: 3′-CCTTCACTGTATTTCCTGCGACAC

For the RT-PCR procedure, cell lines were grown to subconfluence beforeextracting the RNA. Total RNA was extracted using Stat-60™ (Tel-Test™ B)per manufacturer's instructions. First-strand cDNA was prepared from 0.1μg-3 μg of total RNA with the Superscript™ RT kit (Gibco, BRL). PCRamplification of 5 μl of first-strand cDNA was performed in a 50-μl PCRreaction. The above primers were used to amplify first-strand cDNA. Ascontrols, primers corresponding to nucleotide positions 707-729 (sense;5′-GTGGCCCATGCTCTGGCAGAGGG (SEQ ID NO:35)) or 836-859 (sense;5′-GACTGGAGCAAGGTCGTCCTCGCC (SEQ ID NO:36)) and 1048-1071 (anti-sense;5′-GCACCACCCACAAGGAAGCCATCC (SEQ ID NO:37)) of human triosephosphateisomerase (huTPI) (Maquat et al., J. Biol. Chem., 260: 3748-3753 (1985);Brown et al., Mol. Cell. Biol., 5: 1694-1706 (1985)) were used toamplify first-strand cDNA. For mouse triosephosphate isomerase, primerscorresponding to nucleotide positions 433-456 (sense;5′-GACGAAAGGGAAGCCGGCATCACC (SEQ ID NO: 38)) or 457-480 bp (sense;5′-GAGAAGGTCGTGTTCGAGCAAACC (SEQ ID NO: 39)) and 577-600 bp (antisense;5′-CTTCTCGTGTACTTCCTGTGCCTG (SEQ ID NO:40)) or 694-717 bp (antisense;5′-CACGTCAGCTGGCGTTGCCAGCTC (SEQ ID NO:41)) were used for amplification.

Briefly, 4 μCi of (−³²P)CTP (3000 Ci/mmol) was added to each reactionwith 2.5 U of TaKaRa Ex Taq™ (Panvera, Madison, Wis.) and 0.2 μM of eachdNTP. The reactions were amplified in a 480 PCR thermocycler™ (PerkinElmer) using the following conditions: 94° C. for 1 min., 62° C. for 30sec., 72° C. for 1 min, for 18-25 cycles. 5 μl of PCR products wereelectrophoresed on a 6% polyacrylamide gel. The gel was exposed to film.Densitometry measurements were obtained using Alpha Ease Version 3.3a™software (Alpha Innotech Corporation) to quantitate the clone65-specific and clone 320-specific or TPI-specific gene products.

3. Northern Blot Analysis

Adult multiple-tissue Northern blots (Clontech) and the Northern blot ofthe C57MG parent and C57MG/Wnt-1 derivative polyA+RNA (2 μg/lane) werehybridized with a probe designated 65.50mer.2 of nucleotide bases261-310 of FIGS. 1A and 1B:5′-CAACTTCTCGGCCGTGGTGTCTGTAGATGGGCGGCCTGTGAGACTCCAGC (SEQ ID NO:42)generated using the primers noted above. The membranes were washed in0.1×SSC at 55-65° C. and exposed for autoradiography. Blots wererehybridized with a 75-bp synthetic probe from the human actin gene. SeeGodowski et al., Proc. Natl. Acad. Sci. USA, 86: 8083-8087 (1989) for amethod for making a probe with overlapping oligos, which is how theactin probe was prepared.

4. cDNA Library Screening

Clones encoding the full-length mouse clone 65 polypeptide were isolatedby screening RNA library 211: C57MG/Wnt-1 by colony hybridization withthe above probe. The inserts of certain of these clones were subclonedinto pBluescript™ IISK+ and their DNA sequence determined by dideoxy DNAsequencing on both strands.

5. Results

The recently described technique of SSH combines a high subtractionefficiency with an equalized representation of differentially expressedsequences. This method is based on specific PCR reactions that permitexponential amplification of cDNAs which differ in abundance, whereasamplification of sequences of identical abundance in two populations issuppressed. The SSH technique was used herein to isolate genes expressedin a mouse mammary myoepithelial cell transformed with Wnt-1 whoseexpression is reduced or absent in the parental myoepithelial cell. ThepolyA+ RNA extracted from both types of cells was used to synthesizetester and driver cDNAs. The degree of subtraction efficiency wasmonitored by Southern blot analysis of unsubtracted and subtracted PCRproducts using a β-actin probe. No β-actin mRNA was apparent in thesubtracted PCR products, confirming the efficiency of the subtraction.

After RT-PCR and Northern blot analysis were carried out on the initialclone to confirm differential expression, there was found about a 2-foldinduction in the Wnt-1 cell line by Northern blot and a 4.5-foldinduction by RT-PCR. Upon screening of the library, the full-lengthmouse clone 65 was obtained, designated clone 65.11.3. The cDNA formouse clone 65 encodes a novel intracellular protein that is stronglyinduced in the Wnt-1/C57 mg cell line, but is absent, or at very lowlevels, in the parent/C57 mg cells. This clone, 65.11.3, encodes aprotein about 48-60% identical in sequence to members of the Rho familyof small GTPases.

The nucleotide sequence and putative amino acid sequence of mouse clone65 are shown in FIGS. 1A and 1B (SEQ ID NOS:4 and 6, respectively). Thealignment of the human and mouse clone 65 amino acid sequences is shownin FIG. 6 (SEQ ID NOS:3 and 6, respectively). The mouse clone was placedin pRK5E, described above, and deposited with the ATCC. Upontransformation into JM 109 cells, the plasmid renders the cells Ampresistant. Upon digestion with HindIII and NotI, the cells provide amouse insert size of 786 base pairs from the Met codon to the stopcodon. There are 1824 bp upstream of the NotI site encoding mouseheat-stable antigen (HSA) (CD24) fused to the clone 65 insert, which canbe eliminated by digestion with NotI. Because the HSA sequence adjacentto the 5′ end of the gene was removed electronically, there is no 5′sequence for clone 65 upstream of the Met in FIGS. 1A and 1B.

Without being limited to any one theory, the PCR/RT primers may fall inthe 3 UTR of an alternatively spliced clone because the final clone65.11.3 (mouse clone 65) does not have these sequences. A number ofother clones were obtained by screening a C57 mg/Wnt-1 cDNA libraryusing the specific probes described below.

Two subsequent clones, designated as 65.11 and 65.9 and having 2224 and2004 bp respectively, were identified using a probe derived from theoriginal 212 bp clone 65. Their sequences (SEQ ID NOS:12 and 13,respectively) are shown in FIGS. 10 and 11, respectively. Clones 65.11and 65.9 have different 5′ ends, virtually identical 3′ ends, and CArepeats. The 5′ end of clone 65.11 has a CDC-42-like region. The 5′ endof clone 65.9 contains a region with homology to part of a 314-bp EST,AA462-407 (isolated from a mouse mammary gland).

Three other clones were obtained from the same library by screening witha probe derived from the region homologous to CDC-42 of clone 65.11:clone 65.11.1 having 836 bp (SEQ ID NO:14; FIG. 12), the full-lengthmouse clone 65 clone 65.11.3 (SEQ ID NO:15; FIGS. 13A-13B) having 2251bp, described below and the coding region of which is disclosed in FIGS.1A-1B, and sharing a region in the 5′ end with EST AA613604 (isolatedfrom adult mouse placenta), and clone 65.11.6 having 847 bp (SEQ ID NO:16; FIG. 14). Three other clones (65.1, 65.6, and 65.13) were alsoobtained from the primary screen (using a portion of the original clone65 as a probe, one of which (clone 65.1) has a 5′ end similar to that ofclone 65.9. The sequences of these clones (SEQ ID NOS: 17, 18, and 19,respectively) are shown in FIGS. 15, 16, and 17, respectively.

The subsequent clones, which may be splice variants, contain pieces ofthe 3′ end of the initial clone, and/or contain an unusual 5′ end,and/or contain a CDC-42-like end.

Example 2 Isolation of a cDNA clone Encoding Mouse Clone 320

The cDNA for mouse clone 320 was isolated independently by Wnt-1differential screening using the procedure described in Example 1. Theinitial clone isolated was designated clone 320 and had 165 bp. Therewere two clones in this cluster. The clone was at least partiallysequenced as described above and RT-PCR primers were designed asfollows:

320.pcr.top1: (corresponding to bases 2319-2342 of FIGS. 4A-4B)

320.pcr.top1: (corresponding to bases 2319-2341 of FIGS. 4A-4B)5′-GCACACACGCATGGAGGCAAGCTC (SEQ ID NO:43) and 320.pcr.bot1:(corresponding to bases 2423-2446 of FIGS. 4A-4B)3′-ACCACCTCGACATTTGTTCTACCG (SEQ ID NO:44)

RT-PCR and Northern blot procedures were carried out as described inExample 1 to confirm differential expression.

Then four clones encoding at least partial-length mouse clone 320 wereisolated by screening RNA library 211: C57MG/Wnt-1 by colonyhybridization with a probe designated 320.50.mer.1 of nucleotide bases1997-2046 of FIGS. 4A-4B:

(SEQ ID NO:45) 5′-CTCCTGACCTTTGGGGCTGCCACTTCCCAGGACGACCACTGCCTGCC CAC.

The cDNA for mouse clone 320 encodes a novel protein that is stronglyinduced in the Wnt-1/C57 mg cell line, but is absent in the parent/C57mg cells and may be useful in the regulation of cancer cells.

The nucleotide sequence of a consensus sequence made up of all threeclones (SEQ ID NO:7) is shown in FIGS. 4A and 4B. The nucleotidesequence of another clone of mouse clone 320 is shown in FIGS. 5A and 5B(SEQ ID NO:8) and of yet another clone is shown in FIG. 6 (SEQ ID NO:9).The consensus sequence is a mouse clone 320 sequence of 2822 bp havingno obvious or apparent open reading frame, and is probably a partialclone. When RNA from tumors arising in mice in a colony established fromtwo Wnt-1 male transgenic mice (provided by Harold Varmus at NCl) wassubjected to RT-PCR using the above primers, clone 320 was stronglyinduced. A small section of only about 200 bp of the consensus sequencematches a region in the 3′UTR of human Wnt-5A.

The mouse clone was placed in pRK5E, described above, and deposited withthe ATCC. Upon transformation into JM 109 cells, the plasmid renders thecells ampicillin resistant. Upon digestion with BamHI and HindIII, thisprovides a mouse insert size of about 3 kilobase pairs.

Example 3 Isolation of a cDNA Clone Encoding Human Clone 65

To isolate the full-length human clone corresponding to 65.11.3 (mouseclone 65), a human fetal liver cDNA library (Clontech), treated with theSuperScript™ kit using the pRK5E vector as described above, was screenedwith a probe (65.50.mer.2 (SEQ ID NO:42) noted above in Example 1) atlow stringency (20% formamide, 1×SSC, 55° C. wash).

Four clones were identified: 65.1, 65.4, 65.5, and 65.6. The inserts tothese clones were subcloned into pBluescript™ IISK+ and its DNA sequencedetermined by dideoxy DNA sequencing on both strands. A consensussequence from these clones was obtained to give both the nucleotidesequence and putative amino acid sequence for human clone 65. Theconsensus sequence and the derived amino acid sequence are shown inFIGS. 5A and 5B (SEQ ID NOS:1 and 3, respectively). Clone 65.1 (SEQ IDNO:46) starts at nucleotide position 51 and ends at 227 of SEQ ID NO:1.The second clone 65.4 (SEQ ID NO:47) starts at nucleotide position 51and ends at 824 of SEQ ID NO:1. The third clone 65.6 (SEQ ID NO:48)starts at nucleotide position 480 and ends at 1319 of SEQ ID NO:1) inFIGS. 5A-5B. This consensus sequence of FIGS. 5A-5B (SEQ ID NO:1) is 93%homologous to the mouse clone 65 nucleotide sequence of FIG. 1 (SEQ IDNO:4). See FIG. 6. By homology searching, human clone 65, like mouseclone 65, is found to be a member of the Rho, Rac, and CDC42 family.Because of the homology to the Rho and Rac family, these proteins arebelieved to be involved in the upregulation of cancer genes.

The clone was placed in a pRK5E plasmid as described above and depositedwith the ATCC. Upon transformation into JM109 cells, the cells becomeampicillin resistant. Digestion with XhaI and NotI yields an insert sizeof 777 basepairs from the ATG to the stop codon.

Example 4 In Situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis, and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision 1: 169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A (³³-P)UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed-vacuum dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 10 μl each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl RNAsin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. A total of 1.0 μl RQ1DNase was added, followed by incubation at 37° C. for 15 minutes. Atotal of 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, andthe mixture was pipetted onto DE81 paper. The remaining solution wasloaded in a MICROCON-50™ ultrafiltration unit, and spun using program 10(6 minutes). The filtration unit was inverted over a second tube andspun using program 2 (3 minutes). After the final recovery spin, a totalof 100 μl TE was added. Then 1 μl of the final product was pipetted onDE81 paper and counted in 6 ml of BIOFLUOR II™.

The probe was run on a TBE/urea gel. A total of 1-3 μl of the probe or 5μl of RNA Mrk III was added to 3 μl of loading buffer. After heating ona 95° C. heat block for three minutes, the gel was immediately placed onice. The wells of gel were flushed, and the sample was loaded and run at180-250 volts for 45 minutes. The gel was wrapped in plastic wrap(SARAN™ brand) and exposed to XAR film with an intensifying screen in a−70° C. freezer one hour to overnight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer, placed on aluminum trays, andthawed at room temperature for 5 minutes. The trays were placed in a 55°C. incubator for five minutes to reduce condensation. The slides werefixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml s.c. H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNAse-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,and 100% ethanol, 2 minutes each.

B. Pretreatment of paraffin-embedded sections

The slides were deparaffinized, placed in s.c. H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNAse-free RNAse buffer; 37° C., 15 minutes) for human embryo tissue, or8× proteinase K (100 μl in 250 ml RNASE buffer, 37° C., 30 minutes) forformalin tissues. Subsequent rinsing in 0.5×SSC and dehydration wereperformed as described above.

C. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide) The filter paper was saturated. The tissue was coveredwith 50 μl of hybridization buffer (3.75 g dextran sulfate+6 ml s.c.H₂O), vortexed, and heated in the microwave for 2 minutes with the caploosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC, and9 ml s.c. H₂O were added, and the tissue was vortexed well and incubatedat 42° C. for 1-4 hours.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer was added per slide. After vortexing, 50 μl ³³P mixwas added to 50 μl prehybridization on the slide. The slides wereincubated overnight at 55° C.

E. Washes

Washing was done for 2×10 minutes with 2×SSC, EDTA at room temperature(400 ml 20×SSC+16 ml 0.25 M EDTA, V_(f)=4L), followed by RNAseAtreatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml RNAsebuffer=20 μg/ml). The slides were washed 2×10 minutes with 2×SSC, EDTAat room temperature. The stringency wash conditions were as follows: 2hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4L).

F. Oligonucleotides

In situ analysis was performed on DNA sequences disclosed herein. Theoligonucleotides employed for these analyses are as follows.

(1) Clone 65.11 (SEQ ID NO:49) p1: 5′-GGA TTC TAA TAC GAC TCA CTA TAGGGC AGC GTT GAC TCA GAA AAA CC-3′ (SEQ ID NO:50) p2: 5′-CTA TGA AAT TAACCC TCA CTA AAG GGA GCA TAT GAA TTT CAG CCC TAA-3′ (2) clone 320.50 (SEQID NO:51) p3: 5′-GGA TTC TAA TAC GAC TCA CTA TAG GGC ACG CAC ATC TGT TTCCGT TTT-3′ (SEQ ID NO:52) p4: 5′-CTA TGA AAT TAA CCC TCA CTA AAG GGA CCATCC CCG CTC TCT ACC TA-3′

G. Results

In situ analysis was performed on the above DNA sequences disclosedherein. The results from these analyses are as follows.

(1) Clone 65.11

Expression in Mouse tissues: This clone was expressed in developingspinal ganglia of an E15.5 mouse and in the cardiac valve cusps of anadult mouse.

(2) Clone 320.50

Expression in Mouse tissues: This clone was expressed in the pyramidalcell layer of hippocampus and dentate gyrus of an adult mouse brain. Itwas also expressed in the lung, renal medulla, and whisker follicles ofan E15.5 mouse.

Example 5 Use of DNA Encoding Clone 65 and 320 Polypeptides as aHybridization Probe

The following method describes use of a nucleotide sequence encoding aclone 65 or 320 polypeptide as a hybridization probe.

DNA comprising the coding sequence of full-length human clone 65 (asshown in FIGS. 5A and 5B, SEQ ID NO:3), or of mouse clone 65 (as shownin FIGS. 1A and 1B, SEQ ID NO:6), or of full-length mouse clone 320 (thepartial sequences shown in FIGS. 2, 3, and 4; SEQ ID NOS:7, 8, and 9,respectively) is employed as a probe to screen for homologous DNAs (suchas those encoding naturally-occurring variants of these particular clone65 and 320 polypeptides in human tissue cDNA libraries or human tissuegenomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high-stringency conditions. Hybridizationof radiolabeled clone 65-polypeptide- or clone 320-polypeptide-derivedprobe to the filters is performed in a solution of 50% formamide, 5×SSC,0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, phi 6.8,2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours.Washing of the filters is performed in an aqueous solution of 0.1×SSCand 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encoding afull-length, native-sequence clone 65 or 320 polypeptide can then beidentified using standard techniques known in the art.

Example 6 Expression of Clone 65 or 320 Polypeptide in E. coli

This example illustrates preparation of an unglycosylated form of clone65 or 320 polypeptide by recombinant expression in E. coli.

The DNA sequence encoding clone 65 or 320 polypeptide is initiallyamplified using selected PCR primers. The primers should containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector. A variety of expression vectorsmay be employed. An example of a suitable vector is pBR322 (derived fromE. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR-amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode an antibiotic-resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the clone 65-coding or clone 320-codingregion, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates, andantibiotic-resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger-scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After the cells are cultured for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the clone 65 or 320 polypeptide can then be purified using ametal-chelating column under conditions that allow tight binding of theprotein.

Example 7 Expression of Clone 65 or 320 Polypeptide in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof clone 65 or 320 polypeptide by recombinant expression in mammaliancells.

The vector, pRK5E, may be employed as the expression vector. Theappropriate DNA encoding clone 65 or 320 polypeptide is ligated intopRK5E with selected restriction enzymes to allow insertion of the DNAfor clone 65 or 320 polypeptide using ligation methods as described inSambrook et al., supra. The resulting vectors are conveniently referredto generically as pRK5E.clone65 or pRK5E.clone320, respectively, in thegeneral description below.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5E.clone65 or pRK5E.clone320 DNA is mixed with about 1 μg DNAencoding the VA RNA gene (Thimmappaya et al., Cell, 31:543 (1982)) anddissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. Tothis mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mMNaCl, 1.5 mM NaPO₄, and a precipitate is allowed to form for 10 minutesat 25° C. The precipitate is suspended and added to the 293 cells andallowed to settle for about four hours at 37° C. The culture medium isaspirated off and 2 ml of 20% glycerol in phosphate-buffered saline(PBS) is added for 30 seconds. The 293 cells are then washed withserum-free medium, fresh medium is added, and the cells are incubatedfor about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12-hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of the clone 65 or 320 polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum-free medium)and the medium is tested in selected bioassays.

In an alternative technique, the clone 65 or 320 polypeptide may beintroduced into 293 cells transiently using the dextran sulfate methoddescribed by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981).293 cells are grown to maximal density in a spinner flask and 700 μgpRK5E.clone65 or pRK5E.clone320 DNA is added. The cells are firstconcentrated from the spinner flask by centrifugation and washed withPBS. The DNA-dextran precipitate is incubated on the cell pellet forfour hours. The cells are treated with 20% glycerol for 90 seconds,washed with tissue culture medium, and re-introduced into the spinnerflask containing tissue culture medium, 5 μg/ml bovine insulin, and 0.1μg/ml bovine transferrin. After about four days, the conditioned mediaare centrifuged and filtered to remove cells and debris. The samplecontaining expressed clone 65 or 320 polypeptide can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, the clone 65 or 320 polypeptide can be expressedin CHO cells. The pRK5E.clone65 or pRK5E.clone320 can be transfectedinto CHO cells using known reagents such as CaPO₄ or DEAE-dextran. Asdescribed above, the cell cultures can be incubated, and the mediumreplaced with culture medium (alone) or medium containing a radiolabelsuch as ³⁵S-methionine. After determining the presence of the clone 65or 320 polypeptide, the culture medium may be replaced with serum-freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested. The medium containing theexpressed clone 65 or 320 polypeptide can then be concentrated andpurified by any selected method.

Epitope-tagged clone 65 or 320 polypeptide may also be expressed in hostCHO cells. The clone 65 or 320 polypeptide may be subcloned out of thepRK5 vector. Suva et al., Science, 237: 893-896 (1987); EP 307,247published Mar. 15, 1989. The subclone insert can undergo PCR to fusein-frame with a selected epitope tag such as a poly-his tag into abaculovirus expression vector. The poly-his-tagged clone 65 or 320polypeptide insert can then be subcloned into a SV40-driven vectorcontaining a selection marker such as DHFR for selection of stableclones. Finally, the CHO cells can be transfected (as described above)with the SV40-driven vector. Labeling may be performed, as describedabove, to verify expression. The culture medium containing the expressedpoly-His tagged clone 65 or 320 polypeptide can then be concentrated andpurified by any selected method, such as by Ni²⁺-chelate affinitychromatography.

Example 8 Expression of Clone 65 or 320 Polypeptide in Yeast

The following method describes recombinant expression of a clone 65 or320 polypeptide in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of a clone 65 or 320 polypeptide from theADH2/GAPDH promoter. DNA encoding a clone 65 or 320 polypeptide and thepromoter is inserted into suitable restriction enzyme sites in theselected plasmid to direct intracellular expression. For secretion, DNAencoding a clone 65 or 320 polypeptide can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativeclone 65 or clone 320 signal peptide or other mammalian signal peptideor yeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.Recombinant clone 65 or 320 polypeptide can subsequently be isolated andpurified by removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing the clone 65 or 320polypeptide may further be purified using selected column chromatographyresins.

Example 9 Expression of Clone 65 or 320 Polypeptide inBaculovirus-Infected Insect Cells

The following method describes recombinant expression of a clone 65 or320 polypeptide in baculovirus-infected insect cells.

The sequence coding for clone 65 or 320 polypeptide is fused upstream ofan epitope tag contained within a baculovirus expression vector. Suchepitope tags include poly-his tags and immunoglobulin tags (like Fcregions of IgG). A variety of plasmids may be employed, includingplasmids derived from commercially available plasmids such as pVL1393(Novagen). Briefly, the sequence encoding clone 65 or 320 polypeptide orthe desired portion of the coding sequence (such as the sequenceencoding the mature protein if the protein is extracellular) isamplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product is then digested with those selected restrictionenzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al, Baculovirus Expression Vectors: A Laboratory Manual(Oxford: Oxford University Press, 1994).

Expressed poly-his-tagged clone 65 or 320 polypeptide can then bepurified, for example, by Ni²⁺-chelate affinity chromatography asfollows. Extracts are prepared from recombinant virus-infected Sf9 cellsas described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9cells are washed, resuspended in sonication buffer (25 mL HEPES, pH 7.9;12.5 mM MgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), andsonicated twice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8), and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water, and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes non-specifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM imidazolegradient in the secondary wash buffer. One-mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged clone 65 or 320 polypeptide arepooled and dialyzed against loading buffer.

Alternatively, purification of the IgG-tagged (or Fc-tagged) clone 65 or320 polypeptide can be performed using known chromatography techniques,including, for instance, Protein A or protein G column chromatography.

Example 10 Preparation of Antibodies that Bind Clone 65 or 320Polypeptide

1. Polyclonal Antibodies

Polyclonal antisera are generated in female New Zealand White rabbitsagainst murine and human clone 65 polypeptide and against murine clone320 polypeptide. The antigens used are proteins fused with histidine orwith the Fc portion of IgG. Each protein is homogenized with Freund'scomplete adjuvant for the primary injection and with Freund's incompleteadjuvant for all subsequent boosts. For the primary immunization and thefirst boost, 3.3 μg per kg body weight is injected directly into thepopliteal lymph nodes as described in Bennett et al., J. Biol. Chem.,266: 23060-23067 (1991) and “Production of Antibodies by Inoculationinto Lymph Nodes” by Morton Sigel et al. in Methods in Enzymology, Vol.93 (New York: Academic Press, 1983). For all subsequent boosts, 3.3 μgper kg body weight is injected into subcutaneous and intramuscularsites. Injections are done every 3 weeks with bleeds taken on thefollowing two weeks.

2. Monoclonal Antibodies

Techniques for producing monoclonal antibodies that can specificallybind a clone 65 or 320 polypeptide are known in the art and aredescribed, for instance, in Goding, supra. Immunogens that may beemployed include purified clone 65 or 320 polypeptide, fusion proteinscontaining clone 65 or 320 polypeptide, and cells expressing recombinantclone 65 or 320 polypeptide on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the clone 65 or 320 immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1 to 100 micrograms. Alternatively,the immunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectantibodies to clone 65 or 320 polypeptide.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of a clone 65 or 320 polypeptide. Three to four days later,the mice are sacrificed and the spleen cells are harvested. The spleencells are then fused (using 35% PEG) to a selected murine myeloma cellline such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96-well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againsta clone 65 or 320 polypeptide. Determination of “positive” hybridomacells secreting the desired monoclonal antibodies against a clone 65 or320 polypeptide is within the skill in the art. The positive hybridomacells can be injected intraperitoneally into syngeneic Balb/c mice toproduce ascites containing the anti-clone 65 or 320 polypeptidemonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel-exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 11 One Use of Antibodies that Bind Clone 65 or 320 Polypeptide

1. Cell lines

The established human breast tumor cells BT474 and MDA-MB-231 (which areavailable from ATCC) are grown in minimum essential medium (Gibco, GrandIsland, N.Y.) supplemented with 10% heat-inactivated fetal bovine serum(FBS) (Hyclone, Logan, Utah), sodium pyruvate, L-glutamine (2 mM),non-essential amino acids, and 2× vitamin solution and maintained at 37°C. in 5% CO₂. Zhang et al., Invas. & Metas., 11:204-215 (1991); Price etal., Cancer Res., 50:717-721 (1990).

2. Antibodies

Anti-clone 65 or anti-clone 320 monoclonal antibodies that may beprepared as described above are harvested with PBS containing 25 mM EDTAand used to immunize BALB/c mice. The mice are given injections i.p. of10⁷ cells in 0.5 ml PBS on weeks 0, 2, 5 and 7. The mice with antiserathat immunoprecipitated ³²P-labeled Wnt-1 are given i.p. injections of awheat-germ agglutinin-Sepharose (WGA) purified Wnt membrane extract onweeks 9 and 13. This is followed by an i.v. injection of 0.1 ml of theWnt-1 preparation and the splenocytes are fused with mouse myeloma lineX63-Ag8.653. Hybridoma supernatants are screened for Wnt-1 binding byELISA and radioimmunoprecipitation. MOPC-21 (IgG1) (Cappell, Durham,N.C.) is used as an isotype-matched control.

Additionally, the anti-ErbB2 IgG₁κ murine monoclonal antibodies 4D5(ATCC CRL 10463 deposited May 24, 1990) and 7C2, specific for theextracellular domain of ErbB2, may be used with the above antibodies.They are produced as described in Fendly et al., Cancer Research,50:1550-1558 (1990) and WO89/06692.

3. Analysis of Cell Cycle Status and Viability

Cells are simultaneously examined for viability and cell cycle status byflow cytometry on a FACSTAR PLUS™ (Becton Dickinson ImmunocytometrySystems USA, San Jose, Calif.). Breast tumor cells are harvested bywashing the monolayer with PBS, incubating cells in 0.05% trypsin and0.53 mM EDTA (Gibco), and resuspending them in culture medium. The cellsare washed twice with PBS containing 1% FBS and the pellet is incubatedfor 30 minutes on ice with 50 μl of 400 μM 7-aminoactinomycin D (7AAD)(Molecular Probes, Eugene, Oreg.), a vital dye which stains allpermeable cells. Cells are then fixed with 1.0 ml of 0.5%paraformaldehyde in PBS and simultaneously permeabilized and stained for16 hours at 4° C. with 220 μl of 10 μg/ml HOECHST 33342™ dye (also a DNAbinding dye) containing 5% TWEEN 20™.

The data from 1×10⁴ cells are collected and stored using LYSYS II™software and analyzed using PAINT-A-GATE™ software (Becton Dickinson).Darzynkiewica et al., Cytometry, 13:795-808 (1992); Picker et al., J.Immunol., 150:1105-1121 (1993). The viability and percentage of cells ineach stage of the cell cycle are determined on gated single cells using7AAD and Hoechst staining, respectively. (Cell doublets are excluded bypulse analysis of width vs. area of the Hoechst signal.) Cell numbersare determined using a hemocytometer.

4. Affinity of binding to putative receptor

Radioiodinated anti-clone 65 and anti-clone 320 antibodies are preparedby the Iodogen™ method. Fracker et al., Biochem. Biophys. Res. Comm.,80:849-857 (1978). Binding assays are performed using appropriatereceptor-expressing cells cultured in 96-well tissue culture plates(Falcon, Becton Dickinson Labware, Lincoln Park, N.J.). The cells aretrypsinized and seeded in wells of 96-well plates at a density of 10⁴cells/well and allowed to adhere overnight. The monolayers are washedwith cold culture medium supplemented with 0.1% sodium azide and thenincubated in triplicate with 100 μl of serial dilutions of¹²⁵I-anti-clone 65 or clone 320 antibodies in cold culture mediumcontaining 0.1% sodium azide for 4 hours on ice. Non-specific binding isestimated by the preincubation of each sample with a 100-fold molarexcess of nonradioactive antibodies in a total volume of 100 μl. Unboundradioactivity is removed by two washes with cold medium containing 0.1%sodium azide. The cell-associated radioactivity is detected in a gammacounter after solubilization of the cells with 150 μl of 0.1 MNaOH/well. The clone 65 polypeptide and clone 320 polypeptide bindingconstants (K_(d)) and anti-clone 65 and anti-clone 320 antibody bindingaffinities are determined by Scatchard analysis.

It is expected that the antibodies against clone 65 and clone 320polypeptides will affect the growth of these cells. Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va., USA (ATCC):

Material ATCC Dep. No. Deposit Date pRK5E.h.WIG-3.65.4A 209536 Dec. 10,1997 pRK5E.m.WIG-3.65.11.3 209535 Dec. 10, 1997 pRK5E.m.WIG-4.320.9209534 Dec. 10, 1997

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures of the deposits for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the cultures of the depositsto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited materials is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the constructs deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposits of materials herein do not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. Isolated nucleic acid comprising DNA having at least about 800nucleotides and at least about a 70% sequence identity to (a) a DNAmolecule encoding a human clone 65 polypeptide comprising the sequenceof amino acids 1 to 258 of FIGS. 5A and 5B (SEQ ID NO:3), or (b) acomplement of the DNA molecule of (a).
 2. The nucleic acid of claim 1having at least one clone 65 biological activity.
 3. Isolated nucleicacid comprising DNA having at least about 700 nucleotides and at leastabout a 95% sequence identity to (a) a DNA molecule encoding a humanclone 65 polypeptide comprising the sequence of amino acids 1 to 258 ofFIGS. 5A and 5B (SEQ ID NO:3), or (b) a complement of the DNA moleculeof (a).
 4. The nucleic acid of claim 3 comprising DNA encoding a humanclone 65 polypeptide having amino acid residues 1 to 258 of FIGS. 5A and5B (SEQ ID NO:3), or a complement thereof.
 5. Isolated nucleic acidcomprising DNA having at least about 800 nucleotides and at least abouta 70% sequence identity to (a) a DNA molecule encoding a mouse clone 65polypeptide comprising the sequence of amino acids 1 to 261 of FIGS. 1Aand 1B (SEQ ID NO:6), or (b) a complement of the DNA molecule of (a). 6.The nucleic acid of claim 5 comprising DNA having at least about a 85%sequence identity to (a) a DNA molecule encoding a mouse clone 65polypeptide comprising the sequence of amino acids 1 to 261 of FIGS. 1Aand 1B (SEQ ID NO:6), or (b) a complement of the DNA molecule of (a). 7.The nucleic acid of claim 5 comprising DNA encoding a mouse clone 65polypeptide having amino acid residues 1 to 261 of FIGS. 1A and 1B (SEQID NO:6), or a complement thereof.
 8. Isolated nucleic acid comprisingDNA having at least about 800 nucleotides and at least about a 70%sequence identity to (a) a DNA molecule encoding the same full-lengthpolypeptide encoded by the human clone 65 polypeptide cDNA in ATCCDeposit No. 209536 (pRK5E.h.WIG-3.65.4A), or (b) a complement of the DNAmolecule of (a).
 9. Isolated nucleic acid comprising SEQ ID NO:11, 12,13, 14, 15, 16, 17, 18, or
 19. 10. A vector comprising the nucleic acidof claim
 1. 11. A host cell comprising the vector of claim
 10. 12. Aprocess for producing a clone 65 polypeptide comprising culturing thehost cell of claim 11 under conditions suitable for expression of theclone 65 polypeptide and recovering the clone 65 polypeptide from thecell culture.
 13. Isolated nucleic acid having at least about 800nucleotides and produced by hybridizing a test DNA molecule understringent conditions with (a) a DNA molecule encoding a human clone 65polypeptide comprising the sequence of amino acids 1 to 258 of FIGS. 5Aand 5B (SEQ ID NO:3), or (b) a complement of the DNA molecule of (a),and, if the test DNA molecule has at least about a 70% sequence identityto (a) or (b), isolating the test DNA molecule.